Background: The non-selective cation channels TRPV1 (V1) and TRPA1 (A1) are respond to nociceptive stimulus and co-expressed in the dorsal root ganglia (DRG). Capsaicin, a V1 agonist, binds to the transmembrane region of V1, and allyl isothiocyanate (AITC), an A1 agonist, binds to the N-terminus of A1, and activates these channels. It has been suggested that V1 and A1 form a functional heterotetramer in native cells, but the details are unknown. We therefore created their artificial heterotetramers and analyzed whether V1 and A1 could form a functional heterotetoramer.

Methods: A1 and V1 were cloned from rat cDNA, and the A1::V1 tandem(A1::V1) was created by linking them with a linker. We also created a tandem of ∆N A1 and V1 (∆N A1::V1), in which the N-terminus of A1 was shortened. These channels were expressed in HEK293 cells, and the responses of these channels to agonists were examined using the whole-cell patch clamp technique.

Results/Discussion: A1::V1 responded to both capsaicin and AITC. To investigate the contribution of A1 N-terminus to the agonist responsiveness of the A1::V1, we analyzed the function of ∆N A1::V1. ∆N340 A1::V1 (lack of 1st to 8th N-terminal ankyrin domain of A1) was found to be not responsive to both agonists. On the other hand, the ∆N308 A1::V1 (lack of 1st to 7th ankyrin domain of A1) responded to both agonists, suggesting that, the 8th N-terminal ankyrin domain of A1 is required for agonist responsiveness of the A1::V1. 

These results suggest that TRPV1 and TRPA1 constitute heterotetramer and function as ion channels in native cells.

Focal adhesions (FAs) are integrin-based, multiprotein structures that form links between the intracellular actin cytoskeleton and extracellular matrix (ECM). Vinculin is a major FA component and has been proposed to stabilize FAs in a force-dependent manner. While it is known that interaction of vinculin with F-actin is critical for vinculin’s function, how vinculin associates with dynamic actin networks has remained unclear. At the cell periphery, the retrograde actin flow (a continuous centripetal movement of the actin network) is widely observed in adherent cells. By using Sigle-Molecule Speckle (SiMS) microscopy, we found that vinculin in lamellipodia exhibits retrograde flow-associated motion. To clarify how vinculin interacts with the lamellipodial actin network, we examined molecular motions of wild-type vinculin and three kinds of vinculin mutants: constitutively active, lacking F-actin binding, and weak talin binding mutants, respectively. We also observed that vinculin on matured FAs observed by SiMS microscopy exhibits both flowing and stationary fractions. Our findings suggest the following three points: (1) vinculin associates with the lamellipodial actin network mainly via binding to talin. (2) the vinculin-talin complex moves through actin retrograde flow over mature FAs without linkage between F-actin and integrins. (3) Vinculin-talin may form a pre-bound complex in a force-independent manner. These findings, employing direct observation of vinculin and actin at the molecular level, provide new insights into the molecular mechanisms of focal adhesion dynamics.

More than 30% of drugs exert their effects by modulating the activity of G protein-coupled receptors (GPCRs) as agonists or antagonists. To understand the working mechanism of drugs and design new ones, the 3D structural information on therapeutic target proteins is crucial. However, every previous approach to determine the structures of GPCRs requires time-consuming experimental screenings of the expression construct for each target. This process significantly hinders high-throughput structural analysis of GPCRs. Moreover, there is no universal strategy for cryo-EM analysis of GPCRs in both agonist- and antagonist-bound forms.

Here, I present a new method for rapid cryo-EM structure determination of GPCRs, called NOAH (NOvel AI-assisted High-throughput construct screening for structural analysis). NOAH is a program that automatically generates the expression constructs of soluble protein-fused GPCRs suitable for cryo-EM analysis. By employing the NOAH pipeline, we can skip the process of experimental screening, saving a significant amount of time and resources on the project. As a proof-of-concept experiment, I applied this method to three GPCRs and determined not only the antagonist-bound structures but also an agonist-bound structure, demonstrating NOAH’s potential to facilitate GPCR structural biology and drug discovery.

Aging has been progressing rapidly worldwide. Therefore, it is important to investigate age-related differences in disease pathogenesis to develop age-appropriate treatment plan and drug regimen. In this study, we investigated age-related changes in immune function and pathogenesis in a mouse model of asthma. We compared the quantitative and functional changes of various immune cells in untreated or asthma-induced mice between 10-weeks-old (normal group) and 80-weeks-old (aging group) C57BL/6N mice. A mouse model of asthma was generated by repetitive intranasal administration of Dermatophagoides farina. In untreated mice, naive CD4 and CD8 T cells were significantly decreased in the aging group compared with the normal group, whereas effector CD4 and CD8 T cells and regulatory T cells were significantly increased in the aging group compared with the normal group. In a mouse model of asthma, the pathogenesis of asthma was significantly attenuated compared with the normal group based on the result of percutaneous arterial oxygen saturation (SpO₂). In addition, the aging group showed a significant decrease in type 2 innate lymphocytes and eosinophil counts. Our findings indicate that significant downregulation of immune function in the aging group attenuated the pathogenesis of asthma.

The prevalence of food allergy has increased worldwide but the pathogenesis remains undefined and effective treatments has not been established. Transient receptor potential vanilloid 4 (TRPV4), a mechanosensitive nonselective cation channel, is mainly expressed in epithelium of various organs. The present study investigated the role of TRPV4 in the pathogenesis of ovalbumin (OVA)-induced food allergy in mice. TRPV4 was mostly expressed in colonic epithelium. Repeated oral OVA challenge after sensitization induced food allergy, characterized by systemic allergic symptoms, diarrhea, upregulation of Th2-cytokines such as IL-4, IL-5, IL-13, and increase in serum OVA-specific antibodies, but all these responses were significantly augmented in TRPV4-deficient (TRPV4KO) mice compared with wild-type (WT) mice. Infiltration of CD11c-, CD117-, CD4-, and CD170-positive cells in the colon with OVA-induced food allergy was also enhanced in TRPV4KO mice compared with WT mice. Intestinal permeability determined by the FITC-dextran method was significantly increased in TRPV4KO mice compared with WT mice in normal and OVA-induced food allergy. Furthermore, the expression of adherence junction protein E-cadherin, tight junction protein claudin-3 and occludin in the colon was significantly lower in TRPV4KO mice than WT mice in normal and food allergy. These results suggest that epithelial TRPV4 protects OVA-induced food allergy. This response may be accounted for by suppressing the penetration of allergens via maintaining epithelial barrier functions.

Beta caryophyllene (BCP) is one of the sesquiterpenes found abundantly in cannabis as well as other plants such as clove, rosemary, black pepper, and lavender. Past studies have shown this terpene has antioxidant and anti-inflammatory effects, which is believed to be mediated by cannabinoid receptor type 2 (CB2). Recent study showed the attenuation of aortic inflammation by BCP inhalation, which is abolished by the treatment of CB2 antagonist.

CB2 receptor is the Gi/Go type GPCR majorly expressed on the immune-related cells throughout the body. Thus, CB2 has been reported to act as a regulator in the immune system, but not many researches has been conducted to see the therapeutic possibility of CB2 to treat e.g. autoimmune disease by their immune suppression properties. However, since BCP showed inflammatory suppression effect by inhalation, we expected that BCP might be able to attenuate other inflammatory responses, such as allergic diseases through CB2-mediated immune suppression. We therefore examined whether CB2 can suppress allergic responses, particularly by acute inhalation of BCP. The BCP inhalation attenuated sneezing and nose scratch in nasal allergy model mice. Further, the immune cell population of CB2-deficient mice shifted similarly to that of wild-type mice after BCP administration. Altogether, we propose that BCP could be a potential substance to cure allergic diseases.

Background: Quercetin inhibits the histamine release from activated mast cells. Our previous study revealed that the exposure of mast cells to quercetin for 24 hours resulted in an increased release of β-hexosaminidase. Here, we investigated the treatment duration-dependent efficiencies of quercetin on the activations of mast cells.

Methods: Rat basophilic leukemia cells (RBL-2H3 cells) were pre-treated with 1µM quercetin for 0 (control), 1 and 24 hours and the degranulation of mast cells was evaluated. A comprehensive transcriptome analysis was also performed in 1h-, 12h-, and 24h-quercetin-treated RBL-2H3 cells.

Results: A inhibitory effect of quercetin was confirmed in quercetin-treated mast cells with a pre-treatment duration of 1 h. On the other hand, an increase in the activity of mast cells was observed in quercetin-treated mast cells with 24 h pre-treatment period in a dose-dependent manner, compared with those in control cells. The difference in the expression of genes revealed a relationship between the pre-treatment duration of quercetin and cellular activation, such as, but not limited to calcium signaling pathway and FcεRI signaling pathway.

Conclusions: A diversity of functional responses of cultured mast cells to quercetin treatment was observed in the current study. Further study is required for elucidation of treatment duration-dependent efficacy in systemic response of allergic models.

Irritating particulates like PM2.5 cause inflammatory diseases. Such particulates cause phagolysosomal dysfunction of immune cells, resulting in the activation of the Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome, an immune complex that induces cell death accompanied by the release of inflammatory mediators, namely pyroptosis. However, targeting NLRP3 inflammasome-associated responses is insufficient in treating relevant inflammatory diseases, as several particulates like silica particle (SP) also induce NLRP3-independent cell death and release of the inflammatory mediators like interleukin-1(IL-1) α. Therefore, drugs suppressing particulate-induced NLRP3-independent pyroptosis are warranted. In this study, we screened the compounds that can inhibit SP-induced cell death and IL-1α release using a high-content imaging-based system. As a result, we found that several Src family kinase (SFK) inhibitors, including dasatinib, effectively suppressed particulate-induced cell death and IL-1α release. Dasatinib reduced SP-induced phagolysosomal dysfunction. Moreover, dasatinib treatment suppressed the increase in IL-1α levels, and neutrophil count in SP-induced NLRP3-independent acute lung inflammation. In conclusion, dasatinib can inhibit particulate-induced pyroptosis by suppressing the phagolysosomal dysfunction and can be used to treat the relevant disease.

Accumulation of mutated mitochondrial DNA (mtDNA) results in mitochondrial dysfunction. We have previously reported that accumulation of mtDNA mutation increases inflammasome-mediated innate immune response in vitro and in vivo. Emerging evidences suggest that accumulation of maternally-transmitted mtDNA (m-mtDNA) mutation contributes to aggravating ageing. Meanwhile it remains unclear whether m-mtDNA mutation affects immune function in offspring mice. We generated a series of inbred mutant mice by intercrossing of mice heterozygous for the mtDNA mutator allele (PolgAwt/mut), which can generate mice harboring m-mtDNA (PolgAwt1). Bone marrow-derived macrophages (BMDM) from the first generation (N1) of PolgAwt1 (PolgAwt1-N1) display ~1.4 times more mtDNA mutation than cells from the control mice (Polgwt0-N1). However, inflammasome activation in BMDM was comparable between the two groups. There were no significant differences on the number of mtDNA mutation as well as inflammasome activation between cells from PolgAwt1-N2 and PolgAwt0-N2. Although the number of mtDNA mutation in BMDM from PolgAwt1-N3 was comparable to PolgAwt0-N3, inflammasome activation was further increased in BMDM from PolgAwt1-N3, compared to PolgAwt0-N3. These results suggest that m-mtDNA mutation may have an immunomodulatory effect to offspring.

Atmospheric-Pressure Plasma devices with an operational heat close to body temperature have received considerable attention due to their great potential for a variety of biomedical applications, such as acute and chronic wound healing, and regeneration of damaged tissues. Previous studies indicate that reactive oxygen species (ROS)/reactive nitrogen species (RNS) signals indirectly generated by Atmospheric-Pressure Plasma exposure have a positive effect on wound healing. The objective of this study is to investigate the wound healing effects of Atmospheric-Pressure Plasma in a mouse model of thermal injury. Thermal injuries are a major public health problem and cause severe physiological stress. In this project, a mouse model of second-degree burn wound was generated with a solid Al bar (41 g) which preheated in boiling water (80℃) for longer than 1 min on skin in four sites of male C57/BL6 mice for 30 s. The treatments of control gas or Atmospheric-Pressure Plasma were conducted for 15~60 sec for continuous 5 days. Atmospheric-Pressure Plasma was generated by Pidi^® (Sekisui Chemical Co., Ltd.) which is the world's first therapeutic device for animals with gingivitis and halitosis using nitrogen plasma technology. Analysis will be performed on clinical signs, histological evaluations, and gene expression analysis.

Osteoarthritis (OA) is a chronic degenerative disease associated with inflammation and degradation of articular cartilage and is typically considered an age-related disease. In OA patients, persisting pain reduces quality of life. However, the mechanism underlying OA development remain unclear. Recent studies have reported that mitochondrial dysfunction occurs with aging and might contribute to aggravation of inflammation. Hence, we examined whether mitochondrial dysfunction has effects on inflammatory responses in chondrocytes. Primary cultured chondrocytes were prepared from knee articular cartilage of neonatal Wistar rats. Chondrocytes were co-treated with rotenone (Rote), an electron transport complex I inhibitor, and interleukin (IL)-1β. Co-treatment of cultured chondrocytes with Rote and IL-1β potentiated the expression of inflammatory mediators such as matrix metalloproteinase 3, IL-6, and inducible nitric oxide synthase compared to each treatment alone. The potentiation was suppressed by blockade of transforming growth factor beta-activated kinase 1 (TAK1), c-Jun N- terminal kinase (JNK), or nuclear factor-κB (NF-κB). These results suggest that mitochondrial dysfunction potentiates inflammatory responses via the TAK1/JNK/NF-κB pathway in chondrocytes. These responses might contribute to the induction of OA pathogenesis.

Allergic conjunctivitis (AC) causes eyelid swelling, redness, tearing and itching when mast cells release mediators such as histamine (His). We reported 5,6-dihydroxy-8Z,11Z,14Z,17Z-eicosatetraenoic acid (5,6-DiHETE), metabolite of eicosapentaenoic acid, suppressed His-induced inflammation. We investigated the therapeutic effect of 5,6-DiHETE on AC.

Mice were injected i.p. with ragweed pollen (RW) on day 0 and 5 and by eye drop on day 10-14 to induce AC. On day 14, symptoms were scored based on swelling, redness and tearing. In AC, symptom score and tear volume increased 30 minutes after RW. Histological analysis revealed mast cell degranulation and eosinophil infiltration. 300 μg/kg 5,6-DiHETE i.p. just before RW suppressed the increase in symptom score, tearing and histological change. 1 μg 5,6-DiHETE by eye drop also suppressed these symptoms.

To reveal the mechanism, we evaluated the effect on mast cell degranulation in vitro. 1 μM 5,6-DiHETE 15 minutes before antigen treatment suppressed degranulation. We further evaluated the effect on vascular hyperpermeability. 300 μg/kg 5,6-DiHETE i.p. suppressed His-induced tearing and leakage of i.v. injected dye. Orally 600 μg/kg 5,6-DiHETE also suppressed tearing.

In conclusion, 5,6-DiHETE inhibited mast cell degranulation, vascular hyperpermeability and suppressed murine AC.

Depression is a typical psychiatric disorder with a lifetime prevalence rate of approximately 15%. Although the details of the pathogenesis of depression are still unknown, inflammation is considered as the underlying mechanism leading to depression. The chemokine receptor CCR4 is a major regulator of migration of regulatory T cells (Tregs) and Th17 cells. It has been reported that serum levels of CCR4 ligands are increased in patients with depression. However, the involvement of CCR4 in the pathogenesis of depression is still unknown. Here, we investigated the role of CCR4 in depression using a lipopolysaccharide (LPS)-induced depression mouse model. We found that CCR4-deficient mice displayed a significant increase in immobility time in the forced swim test. Flow cytometry analysis revealed that LPS-induced increase in Treg, but not Th17 cell, migration into the brain was decreased in CCR4-deficient mice. Moreover, CCR4 deficiency increased M1 macrophages were increased, while M2 macrophages were decreased in the brain. Similar to CCR4-deficient mice, treatment with selective CCR4 inhibitor decreased Tregs and M2 macrophages in the brain and increased immobility time in the forced swim test. These results suggest that CCR4 suppresses depressive-like behavior via Treg migration into the brain and M2 macrophage polarization.

H₂S generated by three different enzymes including cystathionine-β-synthase (CBS) contributes to somatic and visceral pain by enhancing the activity of Ca_v3.2, an isoform of T-type Ca²⁺ channels (T-channels). Most recently, we have developed KTtp38, a novel derivative of the antipsychotic pimozide, that potently inhibits T-channels, but has little affinity to D₂ receptors. Here, we investigated the effects of KTtp38 on Ca_v3.2-dependent pain, i.e. the somatic and/or colonic pain/hypersensitivity caused by Na₂S, an H₂S donor, or butyrate, and also on the colonic hypersensitivity caused by 2,4,6-trinitrobenzene sulfonic acid (TNBS) in mice. Oral administration of KTtp38 potently suppressed somatic and visceral pain following intraplantar and intracolonic (i.col.) administration of Na₂S, respectively, and the colonic distention hypersensitivity following repeated i.col. butyrate. A single i.col. TNBS caused delayed colonic distention hypersensitivity accompanied by colonic CBS upregulation, which was inhibited by i.p. aminooxyacetic acid, a CBS inhibitor or deletion of Ca_v3.2 gene. Oral or i.p. KTtp38 suppressed the TNBS-induced colonic hypersensitivity. Thus, KTtp38 suppresses Ca_v3.2-dependent somatic and visceral pain, and is considered useful to treat pathological pain involving H₂S and/or Ca_v3.2.

We have demonstrated that high mobility group box 1 (HMGB1) derived from macrophages (Mφ) and anaphylatoxin C5a participate in chemotherapy-induced peripheral neuropathy, which is prevented by thrombomodulin alfa (TMα) that causes thrombin-dependent HMGB1 degradation and thrombin-activatable fibrinolysis inhibitor (TAFI) activation. Thus, we investigated possible involvement of HMGB1 and C5a degradable by the activated TAFI (TAFIa) in the development of diabetic peripheral neuropathy (DPN) in mice with diabetes mellitus (DM). Repeated administration of TMα or an anti-HMGB1-neutralizing antibody prevented DPN development in type 2 DM (T2DM) models, i.e. leptin receptor-deficient db/db mice and high fat diet/low dose streptozotocin (STZ)-induced DM mice, but not in STZ-induced type 1 DM mice. Ethyl pyruvate able to inhibit HMGB1 release from Mφ, minocycline, a Mφ/microglia inhibitor, liposomal clodronate, a Mφ depletor, and DF2593A, a C5a receptor (C5aR) antagonist, reduced or abolished DPN development in the T2DM mice that had upregulation of C5aR, but not HMGB1, in the sciatic nerve. Our data suggest that Mφ-derived HMGB1 and C5a/C5aR participate in DPN development accompanying T2DM, which can be prevented by TMα that causes thrombin-dependent inactivation of HMGB1 and activation of TAFI followed by C5a degradation.

This study aims to understand the connection between chronic low back pain, stress, and depression by developing a mouse model. To develop a low back pain model in mice, Complete Freund's Adjuvant (CFA) was administered to the muscle around lumbar region. To evaluate locomotion and balance ability, the balance beam test was performed. Goal-reaching time was increased from day 14 to day 28 in the CFA-treated group, indicating a long-term decline in locomotor performance. To examine the central influence of low back pain model in the hippocampus, we examined the expression of microglia marker Iba1 and neuorogenic marker NeuroD1 in the dentate gryus (DG) of hippocampus 28 days after CFA administration. Iba1-positive cells were increased, and NeuroD1-positive cells were decreased in the DG of the CFA-treated group, indicating long lasting influence of low back inflammation on the hippocampus. Next, we evaluated the influence of the low back pain model on the responses to chronic stress. Mice were exposed to 10 days of social defeat stress before CFA treatment. Although social avoidance behavior was observed shortly after stress, it dissipated 38 days later in the stress alone group. In contrast, social avoidance behavior was more pronounced in CFA-treated mice than in mice exposed to stress alone. These results demonstrate that CFA-induced low back pain model showed long lasting locomotion and balance inability, histological changes in the hippocampus, and exacerbating the stress response. The developed model in this study can aid in elucidating the neuronal mechanism of low back pain and understanding how low back pain intensifies stress.

Astrocytes are important regulators of CNS functions, including neurotransmission, and play an important role in the etiology of pain. Recently, we have shown that the alcoholism drug disulfiram (DSF) exerts its anticancer effects by directly binding to FROUNT, a protein that promotes signaling through chemokine receptor (CCR)2 and CCR5 expressed on macrophages, and by inhibiting FROUNT function (Nature Communications. 2020: 609). In addition, it has been reported that chemokine receptors including CCR2 and CCR5 are involved in pain regulation. The purpose of this study was to verify the analgesic effects of DSF on various pain models. Formalin model mice were used to evaluate acute nociceptive pain, reserpine model mice were used to evaluate chronic nociceptive pain, and sciatic nerve partial ligation model mice were used to evaluate neuropathic pain. DSF showed analgesic effects on these pain models. In addition, the reduction of pain threshold in reserpine model mice was not observed in FROUNT-deficient mice. And we found that FROUNT-positive cells co-localized with GFAP-positive astrocytes in the dorsal horn of the spinal cord. These results suggest that DSF, an existing treatment for alcohol dependence, may become a new pain treatment with a new mechanism.

Chemotherapy-induced peripheral neuropathy (CIPN) is one of the serious side effects of cancer chemotherapy and significantly reduces the quality of life of patients. However, there are no effective treatments or therapeutic agents for CIPN. Therefore, it is a serious problem in the continuation of anticancer drug therapy. A novel gabapentinoid, mirogabalin (MGB), is used as a treatment for neuropathic pain. In this study, in order to develop a new treatment, we investigated the preventive effects of MGB on mechanical allodynia in a mouse model of vincristine (VCT) -induced peripheral neuropathic pain (VIPN). A single oral administration of MGB dose-dependently inhibited VCT-induced mechanical allodynia. Next, to investigate the action sites of MGB, we evaluated the topical analgesic effects on VIPN model mice.  We found that intrathecal injection suppressed VCT-induced mechanical allodynia but intradermal injection into the footpad did not. Furthermore, intrathecal injection of MGB in healthy mice did not affect locomotor activity. In addition, prophylactic repeated administration of MGB suppressed VCT-induced mechanical allodynia. These results indicate that MGB prophylactically suppresses VIPN by acting on the spinal dorsal horn. MGB may be an effective therapeutic agent for VIPN.

Atopic dermatitis (AD) is the most common inflammatory skin disease with chronic itch. The pathophysiology of AD is complex and multifactorial. The mechanism of chronic itch is poorly understood. Selective serotonin reuptake inhibitors (SSRIs) are used to treatment of depressants. Recently, it is suggested that SSRIs have potential treatments of AD via antipruritic, antimicrobial and immunomodulatory effects. Here, we investigated whether paroxetine, which is the most potent in SSRIs, exerts an antipruritic effect using NC/Nga (NC) mice (Matsuda et al., 1997) and novel AD model mice (FADS mice; Nunomura et al., 2019). The number of scratching behaviors were increased in these model mice as compared with healthy mice. In addition, we examined the effect of paroxetine (10 mg/kg) by single intraperitoneal injection to NC or FADS mice. Paroxetine suppressed scratching behaviors in these mice while H₁ receptor antagonist, terfenadine did not. Furthermore, paroxetine did not affect total distance moved in these mice. These results suggest that SSRIs exert the antipruritic effects for chronic itch by AD.

Few in vitro experimental systems have been optimized for the analysis of the peripheral nervous system (PNS) aimed at elucidating the complex mechanisms underlying the development of peripheral neuropathy. To address this issue, we developed a novel sensory nerve organoid derived from rat embryonic dorsal root ganglion. An innovative advance of present study is that the organoid was composed of independent ganglionic and axonal bundle morphology, which contained unmyelinated C-fibers and A-fibers stereo-myelinated by Schwann cells. After nerve ablation, the axons had almost completely regenerated 2 weeks after injury, indicating that organoids have characteristics specific to the PNS. We also confirmed the mRNA expression of functional molecules (ion channels and receptors), which play important roles in pain transmission, in neuronal cell bodies of organoids. Furthermore, present Ca²⁺ imaging analysis showed that focal application of KCl (30 mM) onto the nerve endings induced Ca²⁺ influx into the neuronal cell bodies. One day after axonal transection, the mRNA expression of stress inducible genes increased in the ganglion. Taken together, this organoid enables real-time evaluation of subtle changes in the PNS, and is considered to be a useful tool for further developing peripheral neuropathy research.

Microglia are involved in induction of chronic pain. In knee osteoarthritis (OA), spinal microglia are activated, but the involvement of brain microglia remains unclear. Therefore, we examined the role of brain microglia in mechanical hypersensitivity in OA.

　The OA model was prepared by administration of monoiodoacetate (MIA) into the left knee joint cavity of ddY male mice . Brain slices prepared after MIA administration were stained with an anti-ionized calcium binding molecular 1 (Iba1) antibody, a marker of microglia. Pain thresholds were measured using von Frey filaments. Cartilage damage was evaluated by Safranin O staining.

　Activation of microglia in the hippocampus was observed from 4 weeks after MIA administration, peaking at 6 weeks and continuing thereafter. The decreased pain threshold and knee joint damage were observed continuously from 2 weeks after MIA administration. Furthermore, at 5 weeks after MIA administration, local administration of clodronate liposome, which deplete microglia, significantly prevented the decreased pain threshold. These results indicate that there is temporal lag between knee joint damage and pain and activation of hippocampal microglia in the OA model mice, and that these cells are involved in persistent pain.

Extracellular ATP (ATPo) is a signaling molecule involved in neurotransmission and neuron-glia signaling and is known to be involved in psychiatric and neurological disorders. However, the exact mechanism of ATP release and the responsible cells under physiological conditions are poorly understood due to the lack of understanding of the spatiotemporal dynamics of ATPo. Using a genetically encoded G protein-coupled receptor activation-based ATPo sensor called GRAB_ATP1.0 , we imaged ATPo near astrocytes in the CA1 region of acute hippocampal slices. Electrical stimulation of the Schaffer collateral resulted in ATPo rise in astrocytes. The ATPo rise was inhibited by TTX, but still remained in the presence of D-APV/CNQX, suggesting that the source of activity-dependent ATP release could be presynaptic sites of neurons. Microglia depletion by PLX5562 prolonged the duration of ATPo rise, suggesting that microglia expressing NTPDase1 rapidly degrade ATPo. Consistently, POM1, a NTPDase inhibitor, increased stimulus-induced ATPo rise. Knockout of IP₃R2, a major source of Ca²⁺ in astrocytes, did not alter the ATPo rise, suggesting　the minor contribution of astrocytic Ca²⁺ to ATPo. Overall data show that neuronal activity induces ATP release from axons or axon terminals, which is negatively regulated by microglia in physiology.

Microglia contribute to synaptic pruning by synaptic engulfment, which is dependent on neuronal activity. Our middle cerebral artery occlusion (MCAO) mice show an upregulation of neuronal activity in the hippocampal dentate gyrus (DG). Thus, we investigated the changes in dendritic spines in DG granule cells following cerebral ischemia and microglial contribution to it. We showed that the number of dendritic spines of DG granule cells in MCAO mice was lower than that in sham mice. However, microglial depletion with CSF1R inhibitor (PLX3397) inhibited the MCAO-induced reduction of the dendritic spine. These results suggest that microglia are involved in the decrease in the number of dendritic spines of DG granule cells following cerebral ischemia.

The immune system of the central nervous system (CNS) consists primarily of innate immune cells. As the tissue-resident macrophages, microglia, which are distributed at equal intervals in the CNS parenchyma, play a pivotal role in the maintenance of tissue homeostasis. During the CNS disease, on the other hand, microglia increase in number and exert their proper function. However, the molecular mechanism by which microglia control their cellular density during homeostasis and perturbation remains unknown. In the present study, we investigated the role of Piezo1, a mechanosensitive ion channel, in the regulation of microglia density. In the brains of mice with specific deletion of Piezo1 in myeloid cells including microglia (Piezo1-KO), the density and the morphology of microglia didn’t differ from those in wild-type controls. However, after pharmacological depletion of microglia with BLZ945, an inhibitor of colony-stimulating factor 1 receptor, Piezo1-KO microglia showed impaired repopulation capacity with less proliferation rate, leading to lower cell density in the brain. Together, these findings suggest that Piezo1 is involved in regulating proper density of microglia following repopulation.

Microglia are resident innate immune cells in the central nervous system (CNS) and play important roles in the development of CNS homeostasis. In several CNS disorders, excessive activation and neurotoxicity of microglia are observed, but the mechanisms that regulate their activation are still unclear. Immune checkpoint molecules (e.g., PD-1, LAG-3) are expressed on activated immune cells and regulate their activation in peripheral immunity. We hypothesized that immune checkpoint molecules could also contribute to the regulation of microglial activation.

First, we analyzed the expression of immune checkpoint molecules in activated microglia using BV2, a mouse microglia cell line. We found that BV2 activated by IFN-γ expressed LAG-3 and the STAT1 signaling pathway was involved in the expression of LAG-3. To investigate the function of LAG-3 in activated microglia, we treated IFN-γ-activated BV2 with an antagonistic anti-LAG-3 antibody to inhibit LAG-3 function. The result showed that the inhibition of LAG-3 increased nitric oxide (NO) production upon IFN-γ activation, indicating that LAG-3 could be suppressing microglial activation.

Our results suggest that activated microglia express LAG-3 and that LAG-3 could function as a negative feedback mechanism to regulate microglial activation.

The proper formation and maintenance of myelin are essential for brain function. It has been found that myelination is regulated in a neuronal activity-dependent manner, resulting from interactions between neurons and oligodendrocytes. Recent reports also suggest that microglia play a crucial role in the formation and maintenance of myelin. However, the specifics of these microglia-myelin interactions remain largely unexplored. To investigate these interactions, we used a mouse cortical slice culture system. This system allowed us to visualize the tripartite relationship between microglia, neurons, and oligodendrocytes using tissue staining and live imaging techniques. This cortical slice culture system was set up by culturing slices from the cortex of 6-day-old mouse brains. Immunostaining of cultured sections with myelin and axon markers revealed that myelin forms on the axons, exhibiting a temporal progression comparable to that seen in mouse cortical sections in vivo. We then examined the impact of neuronal activity on microglia-myelin interactions by modulating neuronal activity using DREADD systems. Our results showed that changes in neuronal activity could have a bidirectional impact on the phagocytosis of myelin by microglia. Our live imaging further disclosed a myelin-like oligodendrocyte structure being phagocytosed by the microglia. In summary, our work has established a cortical slice culture system and suggested that neuronal activity influences the phagocytosis of myelin by microglia.

Various stressors potentiate the rewarding effects of cocaine, which contribute to cocaine craving. However, it remains unclear whether psychosocial stress enhances the rewarding effects of cocaine. To address this issue, we employed a cocaine-conditioned place preference (CPP) paradigm combined with social defeat (SD) exposure and investigated the effects of acute SD stress on cocaine reward in mice. We found that SD stress immediately before the posttest significantly increased cocaine CPP, and systemic blockade of α₁ adrenoceptors (α₁-ARs) suppressed this increase. Fiber photometry recordings with GRAB_NE1m sensors revealed increased noradrenaline (NA) levels in the medial prefrontal cortex (mPFC) during SD. Moreover, the SD stress-induced enhancement of CPP was suppressed by intra-mPFC infusion of an α₁-AR antagonist. In vitro whole-cell recordings showed that silodosin, an α_1A-, but not α_1B- or α_1D-, AR antagonist, inhibited NA-induced depolarizing currents and facilitation of excitatory synaptic transmissions. Consistently, intra-mPFC silodosin infusion suppressed the SD stress-induced CPP enhancement. Additionally, chemogenetic inhibition of mPFC pyramidal cells and intranasal silodosin injection attenuated the CPP enhancement. These findings suggest that NA stimulation of α_1A-ARs and the subsequent activation of mPFC pyramidal cells may contribute to SD stress-induced amplification of the rewarding effects of cocaine, and intranasal silodosin injection may hold therapeutic potential for stress-associated cocaine craving.

Stress potentiates cocaine craving by enhancing the rewarding effects of cocaine, yet the mechanisms underlying this process remain unclear. To address this issue, we examined the role of dopaminergic transmission in the medial prefrontal cortex (mPFC) and basolateral amygdala (BLA), and of reciprocal interaction between these nuclei, using a cocaine conditioned place preference (CPP) paradigm combined with acute social defeat (SD) stress in mice. Brief SD exposure before the posttest enhanced cocaine CPP, which was significantly suppressed by systemic, bilateral intra-mPFC, or bilateral intra-BLA injection of SCH23390 (SCH), a dopamine D1 receptor antagonist. Unilateral mPFC and contralateral BLA injections of SCH also suppressed the stress-induced CPP enhancement, suggesting the dopaminergic modulation of reciprocal glutamatergic connection between the mPFC and BLA. Accordingly, simultaneous injections of SCH into the unilateral mPFC and NBQX, an AMPA receptor antagonist, into the contralateral BLA, inhibited the enhanced CPP. By contrast, simultaneous infusions of NBQX to the unilateral mPFC and SCH to the contralateral BLA failed to affect the CPP enhancement. Moreover, selective inhibition of glutamatergic projections from the mPFC to the BLA with the chemogenetic technique suppressed the enhancement of CPP. These findings suggest that dopaminergic inputs to the mPFC and BLA may modulate glutamatergic transmission from the mPFC to BLA, but not from the BLA to mPFC, which contributes to stress-induced potentiation of cocaine craving.

Chronic social stress affects emotional and cognitive functions and risks psychiatric illnesses such as depression. Rodent studies have shown that chronic social stress induces dendritic atrophy of pyramidal neurons in the medial prefrontal cortex (mPFC), leading to depression-related behaviors. However, how chronic social stress remodels neuronal circuits connected with mPFC neurons is unknown. Here we examined the brain-wide effects of chronic social stress on neuronal projections to the mPFC in mice. Chronic social stress induced behavioral changes, such as social avoidance, decreased reward-directed behavior, and cognitive impairment, which lasted over a month. By unilaterally injecting the retrograde rabies viral vector expressing a fluorescent protein into the mPFC, we visualized the neurons sending axons to the mPFC in more than 150 brain regions. Fluorescently labelled cells in the hippocampus and piriform cortex decreased in stressed mice, while they increased in the orbitofrontal cortex. All these changes were pronounced over a month after the stress. These findings demonstrate that chronic social stress causes long-lasting structural alterations of neuronal projections to the mPFC, which might contribute to post-stress consolidation of depression-related behaviors.

Chronic stress causes depressed mood and cognitive deficits and predisposes to mental illness. Brain metabolic changes are linked to stress pathology, but their molecular mechanism and functional significance remain unknown. In this study, we exposed C57BL/6N male mice to chronic social defeat stress and quantified the metabolites of central metabolic pathways by mass spectrometry imaging. We examined multiple stress-associated brain regions in susceptible mice, which showed stress-induced social avoidance, and resilient mice, which did not. In the medial prefrontal cortex and the hippocampus, chronic stress increased glycolytic metabolites of susceptible mice but not resilient mice. Among susceptible mice, we found a positive correlation between the glycolytic metabolite levels in the medial prefrontal cortex and social avoidance. Knockdown of glucose transporter in this region ameliorated stress-induced depressive-like behavior. In addition, knockdown of the glucose transporter specifically in the hippocampal neurons projecting to the medial prefrontal cortex suppressed stress-induced cognitive dysfunction. These findings suggest that chronic stress induces diverse metabolic alterations across multiple brain regions, with central metabolic changes in the prefrontal cortex and the hippocampus playing a crucial role in stress-related pathology associated with mental illness.

Lifestyle habits have attracted attention as environmental factors of depression.

We analyzed lifestyle habits in high-risk subjects of major depressive disorder (HRMDD). In our analysis of lifestyle habits in HRMDD, we observed elevated sucrose intake.

To investigate how sucrose intake affects stress-induced depression-like behaviors, mice took sucrose liquid freely were subjected to chronic unpredictable mild stress (CUMS). The sucrose intake attenuated CUMS-induced hyperactivity and aggressive behavior but not social deficit. Unexpectedly, the sucrose intake under CUMS impaired recognition memory.

CUMS reduced noradrenaline (NA) tissue levels in the prefrontal cortex. Sucrose intake under CUMS conditions mitigated reduction in NA levels, although it slowed down the turnover of NA, which associated with a decrease in the expression of adrenergic α1 receptors and an increase in the expression of adrenergic α2 receptors.

In this study, it is suggested that increased sucrose intake in HRMDD serves to attenuate stress-induced aggression and hyperactivity. However, this comes with the unintended consequence of impairing cognitive function.These contradictory findings may be attributed to changes observed in NA tissue levels and receptor expression in prefrontal cortex.

Behavioral addiction, defined as an uncontrollable desire to repeat a certain behavior despite negative consequences, has become a social problem. Here, we investigated the role of 5-HT neurotransmission in the nucleus accumbens (NAc) in motivation for wheel running as a model of behavioral addiction in male C57BL/6J mice (> 6 weeks old). Systemic administration of a 5-HT_1A antagonist (WAY100635) increased wheel rotations, while a 5-HT_2A or 5-HT_2C antagonist (volinanserine or SB242084, respectively) decreased them. In the open field test (OFT), WAY100635 or volinanserine did not affect locomotion, but SB242084 increased it. Intra-NAc infusion of SB242084 reduced wheel rotations without altering locomotion in the OFT, whereas intra-NAc infusion of WAY100635 or volinanserine did not affect wheel rotations. Immunohistochemical analysis revealed that wheel running increased the number of c-Fos-positive cells in the NAc, and this increase was reduced by systemic administration of SB242084. Additionally, whole-cell recordings revealed that bath application of a 5-HT_2C receptor agonist (lorcaserin) increased the number of evoked action potentials and spontaneous excitatory postsynaptic currents in NAc neurons. Our results suggest that the activation of the NAc via 5-HT_2C receptor stimulation regulates the motivation for wheel running.

Among antidepressant treatments, electroconvulsive therapy (ECT) is the most efficacious treatment for depression, but the cellular mechanisms underlying the actions of ECT are unknown. Electroconvulsive stimulation (ECS), an animal model of ECT, robustly stimulates hippocampal neurons and gene transcription and enhances adult neurogenesis in the dentate gyrus (DG) of hippocampus. In this study, we focused on CREB, a transcription factor expressed in the hippocampal DG and activated by signals in response to extracellular stimuli. To clarify the role of CREB on increased neurogenesis and expression change in the DG by chronic ECS, we generated adeno associated virus (AAV) expressing GFP and artificial microRNA targeting CREB (miR-CREB) and injected it into the mouse DG. Knockdown (KD) of CREB expression in the DG was confirmed by immunostaining 5 weeks after AAV injection. Eleven times of ECS were administered to CREB KD animals and immunostaining was performed. The repeated ECS significantly increased the number of NeuroD1-positive neural progenitor cells and doubulecortin-positive immature neurons and their dendric elongation in control mice. We found that these cellular changes by repeated ECS were attenuated in the DG of CREB KD animals. We will continue to elucidate whether CREB is involved in these ECS-induced changes in gene expression and whether they correspond to histological changes.

Clozapine (CLZ) is an atypical antipsychotic drug used for treatment-resistant schizophrenia. The target receptors for CLZ, including dopamine D4 and serotonin 5-HT2A receptors, are expressed in various types of neurons with different expression levels. Although cellular and molecular responses by CLZ have been characterized by various studies, neuronal circuit mechanisms underlying the effect of CLZ remain elusive. Thus, it is important to identify characteristics of neurons functionally responsible for the effect of CLZ. For this purpose, we investigated anatomical features of CLZ-responsive neurons in the medial prefrontal cortex (mPFC) by genetic labeling of CLZ-activated neurons and their axons. We found that CLZ-activated neurons in the mPFC have more projections to the mediodorsal thalamus (MD) and the ventromedial thalamus than the basolateral amygdala, whereas vehicle-activated neurons in the mPFC have similar extent of projections to these three regions. We also found that the MD-projecting neurons in the mPFC were activated by CLZ not only in wild-type mice but in a mouse model of schizophrenia by retrograde tracing and immunohistochemistry for c-Fos. These results suggest that the activation of the mPFC-MD circuit is involved, at least partly, in the mechanism for the therapeutic action of CLZ.

Adeno-associated virus (AAV) vectors are powerful tools for cell-type-selective gene delivery to the central nervous system and are promising viral vectors for gene therapy. Despite research on the identification and modification of minimal enhancers and promoters for cell-type-specific gene expression, the application of these enhancers/promoters to AAV often has a trade-off between cell-type specificity and labeling efficiency. Here, we attempted to label targeted neuronal populations specifically and efficiently by optimizing genomic components of AAV. As a result, we established an optimized AAV expression cassette for cell-type-selective and robust gene expression in oxytocinergic, serotonergic and dopaminergic neurons. we also achieved application of oxytocinergic-neuron-selective AAV to labeling the axonal projection patterns on a whole-brain scale in wild-type mice. Our results suggest that the optimization of gene cassettes in AAV vectors can be useful for specific and efficient gene expression toward targeted cell types. These strategies may improve the performance of previously established approaches using AAV vectors with cell-type-specific promoters.

The activity and plasticity of excitatory neurons are regulated in a specific way by local inhibitory neurons. Axo-axonic cells (AACs) are a unique type of inhibitory neurons that primarily form synapses onto the axon initial segment of pyramidal neurons. While their anatomical features have been identified, the functional roles of AACs remain unclear. In this study, we used a specific labeling technique to label AACs in the basolateral amygdala (BLA) and found that they have a crucial role in fear conditioning. By using in vivo calcium imaging of AACs in the BLA, we demonstrated their activation in response to salient stimuli, such as foot shock and reward. Moreover, when AACs were inactivated, the activity of pyramidal neurons increased and fear conditioning was impaired. We discovered that the strength of the inhibitory input from AACs differs between active and non-active neurons during fear conditioning. Additionally, we observed that AACs preferentially receive long-range inputs from the basal forebrain and medial geniculate nucleus. These findings suggest that AACs play a central role in representing salient stimuli and are crucial for regulating BLA activity during memory acquisition.

Memory consolidation depends on the interaction between the hippocampus and the neocortex during slow-wave sleep, and the retrosplenial cortex (RSC) is thought to mediate this interaction. The coupling of hippocampal activity with neocortical slow waves plays a crucial role in memory consolidation, but the dynamics of slow waves in the RSC remain unclear. To investigate whether individual neurons of the RSC exhibit synchronized activity with slow waves, we conducted in vivo whole-cell patch clamp recordings to monitor the membrane potentials of RSC neurons simultaneously with local field potential recordings of slow waves from urethane-anesthetized mice. Among the 40 neurons recorded, 21 exhibited membrane potential dynamics synchronized with slow waves, while 19 exhibited brief and frequent depolarizations without phase locking to neocortical slow waves. Analysis of intrinsic membrane properties revealed that the former were regular spiking neurons, whereas the latter were late spiking neurons. These results suggest that regular spiking neurons, but not late spiking neurons, primarily receive inputs from the neocortex, implying parallel processing by these two cell types in the RSC.

Amyotrophic Lateral Sclerosis (ALS) is a progressive neurodegenerative disease for which no curative treatment is established. Recently, it has been proposed that insufficient energy production due to mitochondrial dysfunction may underlie the ALS pathogenesis, but the precise mechanism remains to be elucidated. Mutant C9ORF72, the most common ALS-causative gene, produces five dipeptide repeat proteins from the abnormally elongated (GGGGCC) sequence. Among them, poly(GR) has been shown to affect mitochondrial function and impair energy production via unknown mechanisms. We focused on the fact that mitochondria have a translation mechanism similar to that of prokaryotes. The newly synthesized mitochondrial proteins can be visualized using the click chemistry technique under cycloheximide treatment because mitochondrial translation is resistant to cycloheximide. We found that poly(GR) strongly inhibited mitochondrial translation, whereas poly(PR), another Arg-rich C9ORF72-dipeptide, did not. We also found that poly(GR) activated mitochondrial UPR (UPRmt) signaling, especially the ATF4/CHOP axis. Therefore, we speculated that aberrant activation of UPRmt by poly(GR) suppresses mitochondrial translation, leading to insufficient mitochondrial energy production and motor neuronal cell death.

Background: Astrocytes constitute about 20~40% of glial cells, making up the central nervous system. Astrocytes usually maintain homeostasis in the extracellular environment, changing their morphology to the activated state in neurodegenerative diseases and releasing inflammatory cytokines. Sphingolipids, one of the lipids, have been reported as a molecule associated with astrocyte activation. Here, we evaluated astrocyte activity by changing sphingomyelin (SM) levels using HASTR/ci35, a conditionally immortalized human astrocyte.

Results＆Discussion: Increasing endogenous SM levels by inhibiting neutral sphingomyelinase and the external addition of SM increased protein and mRNA expression of astrocyte activation markers by IL-1α/TNF-αtreatment. On the other hand, decreasing intercellular SM by the knockdown of sphingomyelin synthase and ceramide transport protein (CERT) reduced protein and mRNA expression of astrocyte activation markers by IL-1α/TNF-α treatment. We analyzed the NF-κB pathway to elucidate how reducing intracellular SM levels suppresses astrocyte activation. We found that CERT knockdown did not alter phospho-IκB, phospho-p65 expression, and p65 nuclear translocation. Interestingly, protein expression of HDAC3, which negatively regulates the NF-κB pathway, was significantly increased. These results suggest that reducing intracellular SM levels suppress astrocyte activation by inhibiting the NF-κB pathway through the induction of HDAC3 expression.

Lipid mediators such as prostaglandins and leukotrienes exacerbated nasal congestion in allergic rhinitis (AR) by increasing blood flow and vascular permeability in nasal mucosa. We here aimed to investigate the effect of a lipoxygenase-metabolite of dihomogammalinolenic acid, 15-hydroxyeicosatrienoic acid (15-HETrE) on functional changes of vasculature, since the previous study showed high level of 15-HETrE was detected in the nasal lavage fluid of AR mouse models. Intranasal administration of 15-HETrE caused abdominal breathing, decreased nasal cavity volume, and increased extravasation of dye injected intravenously in mice. Whole-mount immunostaining revealed that 15-HETrE administration relaxed vessels in nasal mucosa. In ex vivo experiments, the treatment of 15-HETrE relaxed mouse aorta pre-contracted by U46619 in a dose-dependent manner. This 15-HETrE-induced relaxation was inhibited by pre-treatment of prostaglandin D₂ receptor (DP) or prostacyclin receptor (IP) antagonists. Accordingly, the treatment of 15-HETrE on aorta tended to increase the level of intracellular cAMP. Finally, we showed 15-HETrE was detected in patients who complains of AR-related symptoms. These results indicate 15-HETrE can be a novel exacerbating lipid mediator for nasal congestion which stimulates major prostaglandin receptors DP and IP.

The mechanism for myocardial automaticity may differ among different regions of the heart. In this study, we performed fluorescent ion measurements in cardiomyocytes from the sinoatrial node (SAN), the orthotopic pacemaker, and the pulmonary vein (PV), a potential ectopic pacemaker which may cause atrial fibrillation, focusing on the role of the Na⁺/Ca²⁺ exchanger (NCX). Isolated cardiomyocytes from the guinea pig PV and SAN showing automaticity were loaded with the Ca²⁺ indicators Fluo-4 or Indo-1 for high-speed Ca²⁺ imaging. Inhibition of NCX either by SEA0400 or by low Na⁺ solution decreased the Ca²⁺ transient frequency in PV, but not in SAN. The basal cytoplasmic Ca²⁺ concentration, as well as the number of Ca²⁺ sparks between Ca²⁺ transients, were slightly higher in PV than in SAN. Intracellular Na⁺ concentration, measured by a Na⁺ indicator SBFI, was not different between PV and SAN. The equilibrium potential of NCX (E_NCX) was estimated to be less negative in PV than in SAN. In conclusion, NCX is involved in spontaneous activity in PV, but not in SAN. This is probably because the less negative E_NCX and the more negative voltage range for diastolic depolarization in the PV cause a larger driving force for NCX. In the SAN, whose diastolic depolarization largely overlaps with the E_NCX, the role of NCX in automaticity is limited.

Background: Diabetic cardiomyopathy (DCM) is a complication of diabetes that results in pathological cardiac hypertrophy and fibrosis, leading to cardiac dysfunction. DCM also induces cardiac senescence. Epigenetic changes are involved in cellular senescence. However, there is no suitable DCM model that can be created in a short period of time. The purpose of this study is to establish a mouse model of DCM and elucidate the cardiac epigenetic change using the animals.

Methods and Results: Cardiac myoblast H9c2 cells were treated with AGEs (100 or 200 μg/mL). AGEs increased the protein levels of p53 and γ-H2AX as well as the gene expression levels of p21 and p53. Next, we examined a mouse model of type 2 diabetes. C57BL6/N mice (8-week-old, male) were fed a high-fat diet (HFD) and L-NAME (1 g/L) for 4 weeks, followed by daily injections of streptozotocin (STZ) (50 mg/kg/day) for 5 days and sacrificed after another 4 weeks. Picrosirius red staining showed that cardiac fibrosis was increased in L-NAME+HFD+STZ (LHS) mice. LHS mice showed increased gene expression levels of inflammatory cytokines, fibrosis-related genes, and senescence markers. Acetylation and crotonylation levels of histone H3K9 were enhanced in LHS mice.

Conclusion: We established a novel mouse model of DCM, and DCM might be regulated by epigenetic mechanisms.

We have elucidated that the ubiquitin ligase RNF183 is specifically expressed in the kidney, especially in the collecting ducts, which are constantly exposed to hyperosmotic stress. We also identified NKCC1 as a substrate protein of RNF183 using the proximal biotin labeling method with RNF183 fused with the biotin ligase BirA.

In this study, we examined the role of ubiquitination of NKCC1 by RNF183. This analysis was conducted using HEK293 cells expressing RNF183 by the Tet-on system. Our results suggest that RNF183 ubiquitinates NKCC1, promoting its lysosomal degradation. Additionally, in mIMCD cells, which show induced expression of endogenous Rnf183 under hyperosmotic stress, we generated Rnf183-KO cells and examined whether cell death increased or decreased under this stress. The result indicated that the expression of cleaved caspase-3 was elevated in Rnf183-KO cells.

A recent study reported that the expression of RNF183 is upregulated in the colons of patients with inflammatory bowel disease (IBD). Since the molecular mechanisms underlying the development and pathophysiology of IBD are not fully understood, IBD is designated as an intractable disease. Therefore, we plan to analyze the relationship between RNF183 expression and hyperosmotic stress in colon cells and the effects of increased RNF183 expression.

[Background] Podocytes form the essential components of the glomerular filtration barrier and have a critical role in diabetic kidney disease (DKD).  Currently, mounting evidence suggests that glucagon-like peptide 1 receptor agonists (GLP-1RAs), anti-diabetic drugs, have beneficial effects on DKD. However, direct effects of GLP-1RAs on diabetic podocyte injury remain unknown. We investigated whether exendin-4, a GLP-1RA, attenuates hyperglycemia-induced podocytes injury using cultured podocytes and DKD in rat models of type 1 diabetes, and if so mechanism of its beneficial effects. [Methods and Results] Cultured podocytes were exposed to media containing normal (NG; 5 mmol/L) or high glucose (HG; 25 mmol/L) for one week in the presence or absence of exendin-4 (10 nmol/L). HG increased podocytes apoptosis and reduced mRNA expression of novel podocyte markers, synaptopodin and Wilms tumor 1 (WT1).  Exendin-4 reduced podocytes apoptosis and restored these mRNA expression, however, these protective effects were attenuated by the co-treatment with wortmannin, a PI3 kinase inhibitor.  Exendin-4 also preserved Bcl2 and reduced Bax, protein expression. In in vivo study using rat models of streptozotocin-induced type 1 diabetes, exendin-4 suppressed impaired plasma creatinine levels, mesangial expansion, and preserved WT-1-positive podocytes without any changes of plasma glucose levels. [Conclusion] Exendin-4 attenuates hyperglycemia-induced podocyte injury through PI3 kinase signaling pathway, leading to improvement of DKD.

Periodontal disease is a serious problem in the veterinary field, as it is reported that more than 80% of dogs over 6 years of age suffer from periodontal disease. Severe periodontal infection is irreversible; therefore, once the supporting tissues are damaged, there is no possibility of recovery. Therefore, preventive dentistry, such as daily tooth-cleaning and dental gel from early life, at the veterinary hospital as well as at home, is quite important. In this study, we focused on persimmon tannin, a polyphenol extracted from persimmon, and examined the bactericidal, anti-halitosis and anti-inflammatory effects in vitro using Porphyromonas gulae (P. gulae), which is a major contributor to the progression of periodontal disease in dogs. Clinical study in 20 dogs with severe periodontal disease was also conducted by daily oral application of 1% persimmon tannin gel. Whereas persimmon tannins did not alter the growth of P. gulae, significant inhibition of CH₃SH production by P. gulae, and significant inhibition of IL-6, IL-1β and TNFα production by mouse macrophage cell line infected with P. gulae were observed in persimmon tannin treated group. In vitro anti-halitosis, and anti-inflammatory effects of persimmon tannin were confirmed by clinical experiments in dogs with P. gulae-associated periodontal diseases, and one-month oral treatment with 1% persimmon tannin contained dental gel significantly reduced halitosis and P. gulae activity. Our findings suggest that oral treatment with persimmon tannin can be a preventive option for periodontal disease in dogs.

Osteoarthritis (OA) is a chronic inflammatory disease characterized by a decrease in cartilage matrix, disorders of joint movement, and severe pain. Previous studies have suggested that an increase in intracellular Ca²⁺ concentration ([Ca²⁺]­_i) in chondrocytes is associated with OA progression. However, the mechanism underlying this increased [Ca²⁺]_i  is unknown. In the present study, we aimed to elucidate this mechanism and its roles in OA progression. Primary chondrocytes were isolated from C57BL/6 mice, and treated with interleukin (IL)-1β, a major cytokine secreted into synovial fluid during OA. In IL-1β-treated chondrocytes, resting membrane potential was depolarized, and resting [Ca²⁺]_i was increased due to the downregulation of voltage-gated K⁺ channel, Kv1.6. This downregulation of Kv1.6 was also detected in chondrocytes from OA model mice and OA patients. IL-1β induced depolarization of mitochondrial membrane potential (ΔΨm) and cell death. In contrast, overexpression of Kv1.6 in chondrocytes using adenovirus reduced resting [Ca²⁺]­_i, increased ΔΨm, and inhibited cell death. In summary, IL-1β downregulates Kv1.6 and increases resting [Ca²⁺]­_i, resulting in mitochondrial Ca²⁺ overload and subsequent cell death. Our findings may contribute to the understanding of OA pathogenesis and the development of new treatments for OA.

Osteocytes play a crucial role in regulating bone metabolism through their interaction with osteoclasts and osteoblasts. While osteocytes are believed to act as bone mechanosensors, this hypothesis has only been confirmed in vitro and remains unexplored at the in vivo level. In this study, we developed a novel method to visualize osteocyte responses to mechanical loading in live mice using a two-photon excitation microscope.

To facilitate our research, we developed specialized fixtures using an optical 3D printer and securely anchored bone tissue using biocompatible cement. We selected a strain of mice expressing GO-ATeam2, a fluorescence resonance energy transfer (FRET)-based biosensor for ATP, either in the cytosol or mitochondria, as ATP dynamics are recognized as indicative of osteocyte mechanosensing. We obtained fluorescent images from distinct organelles within the osteocytes. Subsequently, we applied static loads along the tibia's long axis using an actuator and monitored the applied force with a load cell. Fluorescent images were captured under various loading conditions. Our investigation into cytosolic ATP dynamics in osteocytes under bone loading, as analyzed through cytosol-localized GO-ATeam2, revealed no significant differences in ratio values at 1N and 3N compared to 0N. However, a noteworthy decrease was observed at 5N and 7N. Similarly, our examination of intramitochondrial ATP dynamics using mitochondria-localized GO-ATeam2 displayed no significant deviations in ratio values across loading conditions ranging from 1N to 7N when compared to 0N.

Our findings suggest that osteocytes do not respond to low loads below 3N. In contrast, excessive loading above 5N results in alterations in the cellular state of osteocytes. Furthermore, our study implies that osteocytes subjected to 5N and 7N mechanical loading exhibit distinct ATP dynamics within osteocyte organelles as a response to mechanical stimuli at the in vivo level.

Nanvuranlat (JPH203, KYT-0353), an inhibitor for L-type amino acid transporter 1 (LAT1; SLC7A5), suppresses the cancer cell proliferation and tumor growth by inhibiting the uptake of large neutral amino acids into cancer cells. Most previous studies have focused on the inhibition of leucine uptake to describe the pharmacological effects of nanvuranlat, mainly because leucine is an essential amino acid that functions as activating signaling molecules of cellular metabolism. In this study, to elucidate the anti-cancer effects of LAT1 inhibitors in more detail, we focused on changes in the intracellular concentrations of all the LAT1 substrates and their importance on cell proliferation. Surprisingly, high-performance liquid chromatography analysis revealed that only three large neutral amino acids were continuously decreased by the treatment with nanvuranlat. Similar changes were commonly observed in multiple cancer cell lines. Culturing the cells in media depleted with each or all of the three amino acids reduced the intracellular amount of corresponding amino acids, partially recapitulating the effects of nanvuranlat on cell proliferation, amino acid signaling, and cell cycle arrest. These findings contribute to understanding the molecular basis underlying the anti-cancer effects of LAT1 inhibitors.

Not published

We have previously identified an anti-cancer compound ACA-28 and its lead compound ACAGT-007a (GT-7) as novel regulators of ERK MAPK signaling using our chemical genetic screen. ACA-28 and GT-7 have unique properties to suppress cell proliferation and induce cell death by further activating ERK in cancer cells with high ERK activity, such as melanoma and pancreatic cancer cells. However, the detailed mechanism of cell death induction by these compounds has not been elucidated. To determine protein targets of ACA-28 relevant for apoptosis induction, we searched for molecules that can bind to ACA-28 and found that BRCA1-associated ATM activator1 (BRAT1), which acts as a DNA damage response protein (DDR protein) upon DNA damage, is a candidate binding protein for ACA-28. We also established a melanoma cell line with acquired resistance to GT-7 (referred to as ACA-R-SK). Interestingly, the expression levels of BRAT1 were significantly higher in ACA-R-SK cells as compared with the original SK-MEL-28 cells. Furthermore, the addition of GT-7 markedly decreased the expression of BRAT1 in both cell lines. Knockdown of BRAT1 by introducing BRAT1 siRNA into the ACA-28 resistant ACA-R-SK cells enhanced cell death induction by GT-7. These results suggest that the down-regulation of BRAT1 may play a key role in the mechanism of cell death induction by GT-7. In this presentation, I will discuss the possible mechanisms of BRAT1 downregulation by GT-7 and the role of BRAT1 in the induction of cell death by GT-7.

References

1) Satoh et al. Identification of ACA-28, a 1’ -acetoxychavicol acetate analogue compound, as a novel modulator of ERK MAPK signaling, which preferentially kills human melanoma cells. Genes Cells 2017, 22, 608-618

2) Khandakar et al. ACAGT-007a, an ERK MAPK Signaling Modulator, in Combination with AKT Signaling Inhibition Induces Apoptosis in KRAS Mutant Pancreatic Cancer T3M4 and MIA-Pa-Ca-2 Cells. Cells 2022,11,702