Amyotrophic lateral sclerosis (ALS) is a motor neuron specific neurodegenerative disease. Accumulation of mutant Cu/Zn-superoxide dismutase (SOD1) protein aggregate in the spinal motor neurons is a common pathological hallmark in several types of ALS animal models and patients. The glymphatic system is a waste clearance system in the central nervous system: the cerebrospinal fluid (CSF) flow through the perivascular space into interstitial spaces and the perivascular localization of aquaporin-4 (AQP4) promote its directional flow and waste clearance. We aimed to show involvement of glymphatic system in disease progression of ALS. We found AQP4 deficiency in SOD1-ALS mice accelerated disease onset and shortened survival period. In addition, abnormal SOD1 protein deposition was increased in SOD1-ALS/AQP4 knockout mice and the clearance of the protein from the spinal cord was slowed in AQP4 knockout mice. Furthermore, we observed AQP4 overexpression and mislocalization and detected glymphatic disfunction in ALS model mice for the first time. We suggest that the aberrant AQP4 distribution in the ALS model mice disrupts directional CSF flow and accelerates accumulation of abnormal proteins in the spinal cord. Our study would provide a new insight on improving the glymphatic system in ALS treatment strategies.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that causes degeneration of upper and lower motor neurons, resulting in muscle weakness and eventual death within 2-5 years after diagnosis. Motor neurons in more than 90 % of the ALS patients contain TAR DNA binding protein-43 (TDP-43) aggregation which is thought to be critical for ALS pathogenesis. However, the molecular mechanism by which this aggregate is formed remains poorly understood.

The gene for valosin-containing protein (VCP), an AAA+ ATPase that unfolds and segregates client proteins from macromolecular complexes or membranes, is mutated in ALS patients. Some of these mutations are demonstrated to alter ATPase activity of VCP. However, how the ATPase activity of VCP contributes to ALS pathogenesis was largely unknown. Here we confirmed that TDP-43 aggregation is formed upon cellular stress conferred by hydrogen peroxide. Treatment of specific VCP inhibitor NMS-873 was found to suppress hydrogen peroxide-induced TDP-43 aggregation without affecting TDP-43 clearance. These results indicate that the ATPase activity of VCP is required for TDP-43 aggregation which is enhanced by ALS-derived mutation in VCP, and thereby VCP ATPase inhibitor such as NMS-873 is a potential therapeutic reagent for ALS patients.

Mutations in sigma-1 receptor (σ₁R) gene are found in ALS. The σ₁R forms oligomers that are regulated by its ligands. However, little is known about the effect of mutations. Here, we transfected motor neuronal NSC-34 cells with σ₁R-mCherry (mCh), σ₁R^E102Q-mCh or untagged forms to assess detergent solubility and subcellular distribution by immunostaining and FRAP. The oligomeric state was assessed using crosslinker. Wildtype σ₁Rs were soluble to detergents, but the mutants were enriched in the insoluble fraction. In the soluble fraction, distribution of mutants appeared in higher sucrose density fractions. Mutants formed aggregates that were co-stained with p62, ubiquitin, and p-PERK, and which had lower recovery in FRAP. Acute treatment with σ₁R agonist SA4503 failed to improve recovery, prolonged treatment (48 h) reduced σ₁R^E102Q-mCh insolubility and inhibited apoptosis. While σ₁R-mCh formed monomers/dimers, σ₁R^E102Q-mCh also formed trimers/tetramers. SA4503 reduced the four types in the insoluble fraction but elevated monomers in the soluble fraction. Co-expression of σ₁R-mCh reduced σ₁R^E102Q insolubility. These results suggest that the agonist and wildtype σ₁R can modify the detergent insolubility, toxicity, and oligomeric states of σ₁R^E102Q. Pharmacological and genetic approaches may be promising to treat σ₁R-related ALS.

Alpha-7 nicotinic receptor (α7 nAChR) is widely distributed in mammalian brain and expressed not only in neurons but also in astrocytes and microglia. Sigma-1 receptor (Sig1R) modulates functions of membrane protein such as ion channel and G-protein coupled receptors via physical interaction. Recent study reported that co-stimulation of both α7 nAChR and Sig1R synergistically induces neuroprotective effect. However, the mechanisms underlying the neuroprotective effects are unclear. Therefore, current study examined whether α7 nAChR can physically interact with Sig1R. HEK293T cells were transfected with plasmids (EYFP, Sig1R-EYFP, Sig1R (1-60)-Halo, Halo-Sig1R (61-223) and α7 nAChR-Myc-Cerulean) by using lipofection. After transfection, the α7 nAChR was immunoprecipitated with Myc antibody. The immunoprecipitants were analyzed by western blotting. The α7 nAChR-Myc-Cerulean can co-precipitated with Sig1R-EYFP, but not EYEP. Moreover, both truncated forms of the Sig1R such as Sig1R (1–60)-Halo and Halo-Sig1R (61–223) can interact with α7 nAChR. Theses results suggested that α7 nAChR can physically interact with some amino regions of Sig1R, and this physical interaction might be important to exert the synergistic effects.

Spinal cord injury (SCI) is a refractory neurodegenerative disease caused by inflammation. M1 microglia induce inflammation, whereas M2 microglia suppress inflammation and show neuroprotective effects. After SCI, M1 cells are predominant compared with M2 cells. Therefore, increasing the predominance of M2 microglia relative to that of M1 microglia is expected to improve SCI. We aimed to evaluate the active constituents of an herbal medicine that induced M2 predominance and investigate the effects of this medicine in SCI model mice. Herbal medicines inducing M2 were screened using cultured microglia. As a result, Polygalae Radix (PR) was found to induce M2 predominance. Oral administration of PR improved motor function in SCI model mice and showed a tendency to increase M2 microglia and protect from axonal degeneration in inured spinal cords. Sibiricose A5 and 3,6′-disinapoyl sucrose were identified in the spinal cord after oral administration of PR. These constituents induced M2 predominance in cultured microglia. Above results indicated that sibiricose A5 and 3,6′-disinapoyl sucrose were transferred to the spinal cord after oral administration of PR, induced M2 predominance of microglia, and improved motor function in spinal cord injured mice. PR may be a promising candidate for the treatment of SCI by inducing M2 predominance.

p38 mitogen-activated protein kinase (MAPK) may contribute to the development of idiopathic pulmonary fibrosis (IPF) through its activation in alveolar epithelial type II cells (AEC II) in response to environmental stresses. We examined whether AEC II-specific　genetically modified p38 activity causes worsening of bleomycin (BLM)-induced pulmonary fibrosis and investigated the potential therapeutic targets for IPF progression by assessing its transcriptome. The three mouse genotypes having different p38 activity in AEC II, MKK6-constitutive active, wild type, and p38-dominant negative, received intratracheal administration of BLM and the lungs were analyzed at 8 days post-instillation, known as an active fibrosis phase. Increased histopathological severity, reduced compliance, and higher collagen content of the lungs correlated with increased p38 activity in AEC II. Transcriptome analysis of them revealed that the differentially expressed genes, upregulated by BLM and associated with upregulation of p38 MAPK pathway, were enriched for functions related to endoplasmic reticulum, extracellular matrix, and immune system. Comparison of our data with a publicly available IPF data determined the target genes involved in IPF progression. These findings indicate that p38 MAPK in AEC II plays an important role in IPF progression.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease characterized by the histopathological pattern of usual interstitial pneumonia and is associated with a high mortality rate. Although the mechanisms underlying IPF pathoaetiologies remain poorly understood, M2 macrophages are believed to play a critical role in pulmonary fibrosis. Several papers have shown that myeloid-derived suppressor cells (MDSCs), which are anti-inflammatory cells as well as M2 macrophages, are increased in the peripheral bloods from IPF patients. In our previous study, we also found that MDSC in bone marrows and lungs from bleomycin (BLM)-treated mice were increased. In the present study, therefore, we examined the role of MDSC in the pathogenesis of pulmonary fibrosis. Administration of anti-Gr-1, a MDSC inhibitor, to BLM-treated mice decreased fibrosis area in the lungs. In contrast, intravenous administration of MDSC enhanced BLM-induced lung fibrosis and expression of α-SMA. These results strongly suggested that MDSCs act as malignant factor promoting the pulmonary fibrosis, as well as M2 macrophage. Now we are investigating the feature of MDSC, using in vitro co-culture system of MDSCs and fibroblasts.

Background: Idiopathic pulmonary fibrosis (IPF) is a lethal lung disease with apoor prognosis. Fibroblast pro- liferation amplifies extracellular matrixdeposition and increases angiogenesis. Vascular endothelial growth factor (VEGF)is one of the most potent angiogenic factors. VEGF interacts with VEGF receptors(VEGFR1 and VEGFR2). A previous study showed that VEGFR1 tyrosine kinase (TK)signaling induced blood flow recovery mediated by bone marrow (BM)-derived stemcells. We hypothesized that VEGFR1-TK signaling might be related topulmonary fibrosis.

Material and methods: Six-week-old male C57Bl/6 wild-type (WT) mice and VEGFR1 TKknockout mice (TKKO mice) were treated with a single intratracheal injection ofbleomycin (BLM; 0.1 μg in 50 μl saline) or vehicle (saline; 50 μl).Lung fibrosis was evaluated by histology, real-time PCR and ELISA forpro-fibrotic factors, and assessment of lung mechanics.

Results: The fibrotic area in the lung and the lung elastance were significantlyreduced in TKKO mice (P < 0.01). The expression of the fibrosis-relatedfactors type I collagen, S100A4, and transforming growth factor (TGF)-β was alsosignificantly reduced in TKKO mice on day 21 after BLM injection. TKKO mice alsohad significantly lower levels of stromal cell-derived factor (SDF)-1 in thelungs and plasma on days 14 and 21 after BLM treatment (P < 0.05). Moreover,the expression of C-X-C chemokine receptor type 7 (CXCR7) and CXCR4, thereceptors for SDF-1, was also suppressed in TKKO mice. Immunohistochemicalanalysis showed that treat- ment with a CXCR4 antibody decreased theaccumulation of VEGFR1+ cells in the lung in WT mice but not in TKKO mice.

Conclusion: These results suggest that VEGFR1 TK signaling promotes BLM-inducedpulmonary fibrosis by ac- tivating the SDF-1/CXCR4 axis in infiltratingVEGFR1+ cells.

Ozone has a strong oxidation effect and has been used for disinfection and sterilization. Currently, frequent disinfection is carried out in medical institutions as an infection control against COVID-19. A regulatory concentration for ozone has been set for the attended environment, and residual ozone must be kept below 0.1 ppm. However, this safety standard is regulated based on healthy people, and the adverse effects of ozone on patients with respiratory disorders such as asthma and pneumonia have to be examined. Therefore, this study aimed to clarify the effect of 0.1 ppm ozone on the development of immune and inflammatory responses in a mouse model of allergic asthma and acute respiratory distress syndrome. The evaluations include pathological analysis of lung tissue, changes in the number of activated immune cells in hilar lymph nodes and bronchoalveolar lavage fluid, serum IgE levels and proinflammatory cytokines, and SpO₂ using pulse oximeter in each mouse model, divided into ozone exposure and control groups.

Our findings provide new information on the adverse effects of low concentration ozone on high-risk patients with a respiratory disorder, including asthma and pneumonia. It suggests that it may serve as a standard for indoor residual concentrations and leakage control measures in ozone disinfection.

Activated T cells are crucial for the development of allergic diseases. We have recently clarified that L-type amino acid transporter 1 (LAT1) plays a functional role in activated T cells. Here, we comparatively investigated the effect of a LAT1 inhibitor, JPH203, on allergic inflammation induced in multiple organs, such as the skin, lungs, and nose of antigen-specific Th2 cell-transferred mice. The local antigen provocation to those mice evoked tissue-specific eosinophilic inflammation, especially accompanied by bronchial and nasal hyperresponsiveness (BHR and NHR) in the lungs and nose, respectively. Antigen-induced ear thickness, BHR, and NHR were significantly suppressed by the administration of JPH203, though the attenuation of eosinophil accumulation was only seen in the skin and nose. The infiltration of antigen-specific T cells determined in the lungs and nasal-associated lymphoid tissue was not affected by the JPH203 treatment. Activation-induced amino acid incorporation, oxidative phosphorylation, glycolysis, cyclin-related protein expression, and resulting cytokine synthesis in Th2 cells were suppressed by JPH203. JPH203 is potentially effective for treating allergic diseases through attenuating the function of activated T cells. However, the mechanisms may not involve the suppression of eosinophil or T cell infiltration in some target organs.

Objective: Pulmonary hypertension (PH) is the most severe complication with Systemic Scleroderma (SSc). In this study, we analyzed the prognostic factors related to PH using a database registered as an intractable disease by the Ministry of Health, Labor, and Welfare (MHLW). 

Methods: The anonymized data were made available to us for analysis (MHLW; No.0708-1; 2010). We used data of patients with SSc registered as a specific disease in 2003-2008 by MHLW. Data cleansing was carried out following the SSc Treatment Guidelines (2012) applied by the MHLW research group. We performed logistic regression for the association between autoantibodies and visceral lesions and prognostic factors analysis for the onset of PH.

Results: With analyzing 22,524 cases registered at 2003-2008, anti-Scl-70 antibody was related with PH (odds ratio 1.54 [95% CI 1.35-1.76]), renal crisis (1.30 [1.00-1.69]), and cardiac conduction disorder (1.15 [1.01-1.31]). Anti-U1-RNP antibody was related to PH (1.46 [1.24-1.73]). We focused on primary and secondary PH and found anti-centromere antibody was related to primary PH (2.01 [1.55-2.62]) while anti-U1-RNP and anti-Scl-70 antibody was related to secondary PH (1.74 [1.43-2.12], 2.11 [1.81-2.45]). As for prognostic factors analysis for the onset of PH after three years with 4,135 cases, the prostaglandin (PG) was identified as a prognostic factor (1.95 [1.17-3.25]), while steroids, immunosuppressants (anti-rheumatic agents), and angiotensin-converting enzyme inhibitors were not identified. 

Conclusion:  The anti-centromere antibody is associated with primary PH, and the anti-Scl-70 antibody is related to secondary PH related to pulmonary fibrosis.  Although PG has been considered to suppress PH's onset, PG is suggested as a prognostic factor for PH.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that is characterized by the irreversible remodeling of the pulmonary artery. Although several PAH drugs have been developed, additional drugs are needed. Rho kinases (ROCKs) are involved in the pathogenesis of PAH, and thus, their inhibitors may prevent the development of PAH. However, the therapeutic benefits of ROCK isoform-specific inhibitors for PAH remain largely unknown. The in vitro and in vivo effects of the ROCK2-specific inhibitor, KD025, were examined herein using pulmonary arterial smooth muscle cells (PASMCs) from idiopathic pulmonary arterial hypertension (IPAH) patients and monocrotaline (MCT)-induced pulmonary hypertensive (PH) rats. The expression of ROCK1 was similar between normal- and IPAH-PASMCs, whereas that of ROCK2 was markedly higher in IPAH-PASMCs than in normal-PASMCs. KD025 inhibited the accelerated proliferation of IPAH-PASMCs in a concentration-dependent manner (IC50 = 289 nM). Accelerated proliferation was also reduced by the siRNA knockdown of ROCK2. In MCT-PH rats, the expression of ROCK2 was up-regulated in PASMCs. Elevated right ventricular systolic pressure in MCT-PH rats was attenuated by KD025 (1 mg/kg/day). These results strongly suggest that enhanced ROCK2 signaling is involved in the pathogenic mechanism underlying the development of PAH, including accelerated PASMC proliferation and vascular remodeling in patients with PAH. Therefore, ROCK2 may be a novel therapeutic target for the treatment of PAH.

Tumor necrosis factor (TNF)-α is a major pro-inflammatory cytokine involved in the pathogenesis of several ocular diseases. Previous studies have shown that capillary degeneration and inflammatory responses occur in the retina following an intravitreal injection of N-methyl-D-aspartic acid (NMDA) in rats. In this study, we aimed to determine the role of TNF-α in capillary degeneration in an NMDA-induced retinal injury model of neonatal rats. Intravitreal injection of NMDA (200 nmol) was performed on postnatal day (P) 7. We examined 1) changes in protein level and distribution of TNF-α and 2) effects of TNF-α neutralizing antibody (anti-TNF-α Ab) on the capillary degeneration in retinas of NMDA-injected eyes. The protein level of TNF-α increased 2 days (i.e., P9) after NMDA injection, and the enhanced immunoreactivity for TNF-α was observed in the ganglion cell layer. Intravitreal injection of anti-TNF-α Ab (0.1 μg) at 2 days after NMDA injection suppressed retinal capillary degeneration. These results suggest that TNF-α plays an important role in capillary degeneration following neurotoxicity in the retina.

Fluorescence imaging has made it possible to observe biomolecular distribution inside living cells. While a variety of fluorescent proteins are widely used, imaging tools using small-molecular organic fluorophores has attracted much attention due to their superior properties such as photostability. We have developed DeQODE chemical tag technology for genetically targeted fluorescence imaging. In this labeling system, a small-molecular QODE probe consisting of an organic fluorophore and a dinitrophenyl (DNP) quencher is almost non-fluorescent but becomes highly fluorescent upon binding to an anti-DNP single-chain antibody, DeQODE tag, expressed in cells. Since the probe reversibly binds to the tag protein, fluorescent signal can be continuously obtained, even if the bound probe photobleaches by strong excitation usually used in super-resolution imaging, by an exchange reaction in which another intact probe binds to the tag after the photobleaced probe is dissociated. We applied DeQODE chemical tag to live-cell STED microscopy, and achieved continuous super-resolution imaging of synaptic protein Homer in rat hippocampal neurons. This result clearly showed that DeQODE chemical tag technology is a powerful tool for super-resolution imaging by avoiding the problem of photobleaching.

Pancreatic β-cells release insulin in a Ca²⁺-dependent pulsatile manner to control blood glucose levels. Spatiotemporal kinetics and function of Ca²⁺ signaling in β-cells have been studied by experiments using in vitro and ex vivo preparations. However, in vivo analysis remains challenging. While β-cell activities in vivo are under the influence of the autonomic nervous system, hormones and other bioactive substances, Ca²⁺ activities of β-cells under physiological conditions have not been clarified. We here report a method to analyze in vivo β-cell Ca²⁺ signals using a transgenic mouse line expressing a genetically encoded ratiometric Ca²⁺ indicator, YC-Nano50. Using the method, we visualized β-cell Ca²⁺ signals in laparotomized mice under anesthesia, and observed synchronized Ca²⁺ oscillations in β-cells within individual islets. Furthermore, we succeeded in monitoring Ca²⁺ activities in multiple islets simultaneously, which may clarify the basis for a pulsatile insulin secretion. Further studies using current method is expected to gain deeper insight in insulin secretion mechanism and the etiology of diabetes.

Stochastic and proliferative events initiating from a single cell can disrupt homeostatic balance and lead to fatal disease such as cancer metastasis. To overcome metastasis, it is necessary to detect and quantify sparsely-distributed metastatic cells throughout the body in the early stages. Here we demonstrate that CUBIC (clear, unobstructed brain/body imaging cocktails and computational analysis)-based cancer (CUBIC-Cancer) analysis with a refractive-indices (RI) optimized protocol enables comprehensive cancer cell profiling in whole body and organs. CUBIC-Cancer analysis is applicable to a dozen mouse models using several cancer cells and spatio-temporal quantification of metastatic cancer progression at single-cell resolution. CUBIC-Cancer analysis is applicable to profiling of the remodeling of the tumor microenvironment. The scalable analytical pipeline with these three modalities would contribute to overcome incurable metastatic diseases.

Therapeutic drugs have been classified based on pharmacodynamics and disease indications. However, it has gradually been revealed that profiling of side effects can be used to classify therapeutic drugs and to find novel disease indications of drugs. In this study, we generated multidimensional vectors for each therapeutic drug based on the cosine similarity of side effects in Adverse Event Open Learning through Universal Standardization (AEOLUS), a standardized version of the US FDA Adverse Event Reporting System. Density-based spatial clustering was applied to the multidimensional vectors based on the side effects in AEOLUS. By comparing these clusters, we were able to identify several sets of therapeutic drugs, including a few sets comprising of therapeutic drugs with different pharmacodynamics. These findings suggest that clustering therapeutic drugs based on similarities of indications and side effects reported in public databases can be useful to find new functions of therapeutic drugs.

Increased commercial demand for monoclonal antibodies has resulted in an urgent need to establish alternative production systems. We previously developed a transgenic chicken bioreactor system that efficiently produced human cytokines in egg whites from genome-edited transgenic chickens. Here, we report on the application of this system to monoclonal antibody production. The genes encoding heavy and light chains of humanized anti-HER2 mAb linked by a 2A peptide sequence were integrated into the chicken ovalbumin locus using a CRISPR/Cas9 protocol. The knock-in hens produced a fully assembled humanized monoclonal antibody in their eggs. The mAb expression level in the egg white was 1.5–2.0 mg/mL by ELISA. Furthermore, the antigen binding affinity of the anti-HER2 mAb in egg white was estimated to be equal to that of the therapeutic anti-HER2 mAb (trastuzumab). In addition, antigen-specific binding by the egg white mAb was demonstrated by immunofluorescence against HER2 positive and negative cells.　These results indicate that the chicken bioreactor system can efficiently produce mAbs with antigen binding capacity and has the potential to be an alternative production system for commercial mAbs.

【Backgrounds】

Mammary gland tumors are the third most common tumor in cats, and in contrast to the canine mammary gland tumors, which are mostly benign, the malignancy of the cancer is high, and the survival rate is low. Besides postoperative recurrence of the cancer is more likely to occur, resulting in a lower quality of life for the affected cats and the burden of treatment costs on the owners due to long-term treatment. Because there are few cell lines available for feline mammary tumors, little research has been done to improve their treatment. The organoid culture method can reproduce cancer epithelial structures in vivo on a three-dimensional culture dish and is considered to be a breakthrough in basic and clinical cancer research as a way to maintain cell diversity and stem cell nature. Therefore, the aim of this study was to establish a feline mammary tumor organoid culture method using mammary tumor-affected cats.

【Methods and Results】

Tumor tissues surgically removed from mammary tumor diseased cats were used for organoid culture. The generated organoids showed different histological features and reproduced the epithelial structure of the original tumor tissue. The response to anti-cancer drugs was also different in each organoid. In addition, strains with high HER2 expression showed reduced cell viability at lower concentrations with lapatinib treatment.

【Conclusion】

These findings revealed that feline mammary tumor organoids might become a useful tool to investigate the mechanisms of pathogenesis and treatment of feline mammary tumors.

Tumor blood vessel structure is different from normal blood vessel features in terms of short lumen diameter, serpentine course, irregular sprouting and poor tight junction formation. These phenomena lead to form leaky tumor vessels with low blood flow. These tumor microenvironments (TME) lead to hypoxia. The TME polarizes tumor-infiltrating macrophages towards tumor supportive phenotype. Macrophage-abundant tumors are highly malignant and are the cause of poor prognosis and therapeutic resistance.

In this study, we have used Lewis lung carcinoma (LLC) syngeneic tumor mouse models, which existed abundant macrophages in tumor tissues. These cells were subcutaneously transplanted into the right flank of mice. Mice were treated with prolyl hydroxylase (PHD) inhibitors intraperitoneally at day10 after tumor transplantation. Once every other day, tumors were measured in two dimensions and the tumor tissue volume was calculated. Tumor tissues were collected at day16 and analyzed tumor vessels and immune cells by immunofluorescence staining.

PHD inhibitors treatment induced tumor blood vessel reconstitution and normalization in tumor tissues, these results led to increase perfusion and oxygenation. macrophages contributed to blood vessel normalization. In this study, we characterized macrophage function and subsets which can be altered phenotype after PHD inhibitors treatment. Our results imply that the PHD inhibitors could promote the anti-tumor potential of macrophages to improve malignant cancer therapy.

We reported that differentiation-inducing factor-1 (DIF-1) inhibited the proliferation of various cancer cells including malignant melanoma and that DIF-1 prevented lung colony formation in a mouse model of metastatic melanoma. However, the mechanisms of this action remain to be elucidated. In the present study, we investigated the anti-metastatic effects of DIF-1 in human BRAF^V600E mutation bearing malignant melanoma A2058 cells. Activities of cell migration and invasion were measured by the wound healing assay and cell invasion assay, respectively. Activities of cell adhesion to extracellular matrix (ECM) were measured by using ECM-coated plate. Expression levels of signaling molecules were measured by Western blotting. DIF-1 suppressed the phosphorylation levels of signal transducer and activator of transcription 3 and subsequently reduced a variety of genes related to mesenchymal features such as matrix metalloproteinase-2, vimentin, N-cadherin and twist. DIF-1 also suppressed a variety of genes related to cell-matrix adhesion such as focal adhesion kinase, Src, paxillin. Consequently, DIF-1 inhibited cell migration, invasion and adhesion to ECM. These results suggested the possibility that DIF-1 suppresses the detachment of malignant melanoma cells from the primary tumor by inhibiting mesenchymal features which exhibit aggressive and metastatic phenotypes.

Purpose: CD44 is widely expressed on the surface of most tissues and all hematopoietic cells. CD44 plays important roles in cell proliferation, adhesion, and migration. Previously, we developed an anti-CD44 monoclonal antibody, C₄₄Mab-5 (IgG₁, kappa) by immunizing mice with CD44‑overexpressing Chinese hamster ovary (CHO)-K1 cells. In this study, we converted the mouse IgG₁ subclass antibody C₄₄Mab‑5 into an IgG_2a subclass antibody, 5‑mG_2a, and further produced a defucosylated version, 5‑mG_2a‑f and investigate its antitumor activities against oral cancers.

Methods: To generate 5‑mG_2a‑f, appropriate V_H cDNA of mouse C₄₄Mab‑5 and C_H of mouse IgG_2a were subcloned into pCAG‑Neo vector, and light chain of C₄₄Mab‑5 was subcloned into pCAG‑Ble vector. Vectors were transfected into BINDS‑09 (FUT8‑deficient ExpiCHO‑S cells) using the ExpiCHO Expression System. Mouse xenograft models of HSC-2 and SAS (human oral cancer cell lines) were used for examining the antitumor activity.

Results: 5-mG_2a-f demonstrated a sensitive and specific reaction against oral cancer cells in flow cytometry and immunohistochemical analyses. The sensitivity of 5‑mG_2a‑f was similar with that of C₄₄Mab‑5. In vitro analysis demonstrated that 5‑mG_2a‑f showed moderate ADCC and CDC activities against HSC-2 and SAS. 5-mG_2a-f significantly reduced tumor development in HSC-2 and SAS xenografts in comparison to control mouse IgG.

Conclusion: 5‑mG_2a‑f may be a useful antibody-based therapy for patients with CD44-expressing oral cancers.

Glioblastoma (GBM), the most malignant type of primary brain tumor, has a very poor prognosis. Glioma stem cells (GSCs) play a key role in tumor initiation and progression. Cyclin-dependent kinase 8 (CDK8), which belongs to the transcription-related CDK family, is considered both an oncogene and a tumor suppressor. However, the functional role and underlying mechanisms of CDK8 expressed in GSCs on gliomagenesis is still poorly understood both in vitro and in vivo. Disruption of CDK8 by shRNA resulted in an attenuation of the self-renewal potential and tumorgenicity of patient-derived GSCs, which can be significantly rescued by the overexpression of MYC, a stem cell transcription factor. Moreover, the pharmacological inhibition by CDK8 inhibitor significantly repressed the self-renewal potential and tumorgenicity of GSCs. Bioinformatics analyses have revealed thatCDK8 expression was significantly higher in human GBM tissues compared with normal brain tissues, and its expression was positively correlated with stem cell markers including MYC and SOX2 in human GBM specimens. Additionally, CDK8 expression is associated with poor survival in GBM patients. These findings highlight the importance of the CDK8-c-MYC axis in maintaining stemness and tumorigenicity in GSCs, indicating that targeting GSCs through CDK8 inhibition could be a promising strategy against GBM.

Introduction:  Cylindromatosis (CYLD) regulates various cell signaling pathways by acting as a deubiquitinating enzyme. Although it was shown that loss of CYLD expression was associated with poor prognosis in glioblastoma multiforme (GBM), the biological roles of CYLD in GBM remain unknown. Here, we elucidated the biological significance of CYLD in the malignant characteristics of GBM.

Methods:  To assess the biological significance of CYLD in GBM cell line (U251MG), we performed CYLD knocked-down by CYLD-specific siRNA and CYLD overexpression by wild-type CYLD plasmid. Next, we evaluated cell migration by cell migration assay, cell morphological change by endothelial tube formation assay, and cancer stem-like characters by sphere formation assay.

Results: In U251MG, CYLD knocked-down significantly promoted cell migration, while CYLD overexpression suppressed it. CYLD knocked-down also induced cell morphological change like vascular mimicry (VM), whereas CYLD overexpression inhibited it. Moreover, CYLD knocked-down promoted cancer stem-like characters, while CYLD overexpression suppressed it.

Conclusion: Loss of CYLD expression may be associated with the malignant characters, such as, invasion, VM, and cancer stem-like characters of GBM.

The effect of angiotensin II (Ang II) on metastatic lesion formation and tumor microenvironment were analyzed by in-vivo and in-vitro experiments. Triple negative breast cancer 4T1 cells constitutively expressing luciferase were subcutaneously injected into mammary fat pad of BALB/c mice. Ang II was administered using osmotic pump and valsartan (Val) was administered orally once a day. Four weeks after cell injection, primary tumor was removed for analysis. Lung metastasis was also evaluated by micro-CT imaging and the measurement of luciferase activity. Ang II infusion significantly accelerated the growth of primary tumor and lung metastatic lesion formation, however Val treatment significantly attenuated them. Snail and c-Myc protein expressions were significantly increased in primary tumors of Ang II-infused mice but Val treatment reversed them. However, in vitro, Ang II did not affect proliferation, migration, invasion or protein expressions of 4T1 cells. In contrast, Ang II significantly increased Snail and c-Myc protein expressions when 4T1 cells were co-cultured with dermal fibroblasts. These results indicate that Ang II affects tumor microenvironment to accelerate the tumor growth and metastatic lesion formation.

(Pro)renin receptor [(P)RR] has role in various diseases, such as cardiovascular and renal disorders and cancer. Aberrant (P)RR expression is prevalent in pancreatic ductal adenocarcinoma (PDAC) which is the most common pancreatic cancer. Here we show whether aberrant expression of (P)RR directly leads to genomic instability in human pancreatic ductal epithelial (HPDE) cells. (P)RR-expressing HPDE cells show obvious cellular atypia. Whole genome sequencing reveals that aberrant (P)RR expression induces large numbers of point mutations and structural variations at the genome level. (P)RR-expressing cell population exhibits tumour-forming ability, showing both atypical nuclei characterised by distinctive nuclear bodies and the chromosomal abnormalities. (P)RR overexpression upregulates SWItch/Sucrose Non-Fermentable (SWI/SNF)-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 5 (SMARCA5) through a direct molecular interaction, which results in the failure of several genomic stability pathways. These data reveal that aberrant (P)RR expression contributes to the early carcinogenesis of PDAC.

Background: Cisplatin is widely used as an anti-tumor drug for the treatment of solid tumors. Unfortunately, it causes nephrotoxicity as a critical side effect, limiting its use, given that no preventive drug against cisplatin-induced nephrotoxicity (CIN) is currently available. In the present study, we searched and identified candidate drugs for preventing CIN

Methods: We used a database of medical big data for the screening of candidate drugs for the prevention of CIN. Based on the results of the analysis of medical big data, we evaluated the actual efficacy of DPH via in vitro and in vivo experiments in culture cells and a mouse model.

Results: We identified that a previously developed drug, diphenhydramine (DPH), may provide a novel treatment for CIN by the analysis of medical big data. DPH inhibited cisplatin-induced cell death in renal proximal tubular cells. Mice administered cisplatin developed kidney injury with renal dysfunction, augmented oxidative stress, increased apoptosis, and elevated inflammatory cytokines; however, most of these symptoms were suppressed by treatment with DPH. Additionally, the renal concentration of cisplatin was attenuated in DPH-treated mice. Importantly, DPH did not i interfere with its anti-tumor effect in any of the in vitro or in vivo experiments. Moreover, a retrospective clinical study showed that patients with malignant cancer who had used DPH before cisplatin treatment exhibited less acute kidney injury.

Conclusion: DPH may be a preventive medicine against CIN.

Microbial imbalance (dysbiosis) is closely linked to several diseases including cardiovascular diseases. Gram-positive Streptococcus genus is reported to be increased in feces of spontaneous hypertensive rat (SHR) with increased intestinal permeability. However, the mechanisms of the dysbiosis induced high blood pressure remains unknown. In this study, we pharmacologically examined the effect of streptolysin O (SLO), a streptococcal pyrogenic exotoxin, on vascular functions (1. Relaxation 2. Contraction).

(1) Relaxation results: In aortas isolated from Wistar rat, in vitro treatment with SLO (1-100 ng/ml, 30 min) impaired acetylcholine (ACh)-induced endothelial dependent relaxation in a dose-dependent manner (n=6, p&lt;0.05). In contrast, SLO did not change sodium nitroprusside-induced endothelial independent relaxation (n=4). Endothelial dysfunction caused by SLO was attenuated by pan protein kinase C (PKC) inhibitor (Ro 31-8222, n=6, p&lt;0.05), PKCb inhibitor (LY 333531, n=5, p&lt;0.05) or selective PKCb2 inhibitor (CGP53353, n=5, p&lt;0.05). In vivo treatment of Wistar rat with SLO (0.1-10 ng/ml) blunted ACh-induced blood pressure reduction (n=4, p&lt;0.05).

(2) Contraction results: In vitro treatment of aortas with SLO (1-100 ng/ml, 30 min) did not change contractile responses to noradrenaline (NA), serotonin (5-HT; n=4). Ex vivo treatment with SLO (10 ng/ml, 24 hr) did not change contractile response to NA or 5-HT (n=6).

We conclude that SLO causes endothelial dysfunction through PKCb signaling and might contribute to the development of hypertension.

Aim: Prolyl-hydroxylase domain protein inhibitor (HIF-PHD inhibitor) is developed for the treatment of renal anemia. Patients treated with the HIF-PHD inhibitor often have complications such as renal dysfunction and high blood pressure. Renal damage often disrupts water and electrolytes regulation, and could retain salt, which further disrupts the homeostasis. In this study, we investigated the effect of a HIF-PHD inhibitor, molidustat, on salt excretion and distribution in the rats with subtotal nephrectomy-induced nephron loss.

Methods: Male Wistar rats were performed with 5/6 nephrectomy. After confirming the blood pressure elevation (>150 mmHg), the rats were divided into four groups and given either vehicle, molidustat (2.5 and 5 mg/kg/day, p.o.) or erythropoietin (100 IU/kg, twice a week, s.c.) for 1 week.

Results: Molidustat treatment was initiated after 4 weeks of 5/6 nephrectomy. After one week of treatment, molidustat did not improve blood cell volume or blood pressure. Molidustat had no effect on urinary sodium excretion or concentration after the acute oral salt loading (1 g/kg). Furthermore, tissue accumulation levels of sodium, potassium, and water in the skin, carcass and bones were not affected by molidustat.

Conclusion: Molidustat affected neither excretion nor accumulation of salt in the rats model of nephron loss.

We previously reported that endogenous glucocorticoid elevation induces skin-specific sodium and water loss. In the present study, we examined the effect of exogenous glucocorticoid administration on body sodium and water balance in mice by using dexamethasone, a potent corticosteroid with predominantly glucocorticoid actions. At 24 hours after dexamethasone injection (1 mg/kg/day, s.c.), dexamethasone-treated mice exhibited the decrease in skin sodium and water content with the increase in urinary sodium excretion and urine volume, suggesting that acute dexamethasone injection induces skin sodium and water loss accompanied by renal sodium and water excretion. On the other hand, continuous administration of dexamethasone for 3 weeks also reduced skin sodium and water content although urinary sodium excretion and urine volume were not significantly altered. This skin-specific sodium loss independently of renal function was associate with increased lymphatic vessel endothelial hyaluronan receptor-1 mRNA levels in the skin, suggesting that expanded skin lymph vessels enhanced lymphatic sodium and water clearance in continuous dexamethasone-treated mice. Our findings confirm that fluid homeostasis is maintained by kidney and skin barriers. Glucocorticoid induces skin-specific sodium osmolyte loss via the lymphatic clearance system, which causes skin dehydration.

Hypothesis Interstitial cystitis (IC) is a chronic inflammatorydisease characterized by pain in the bladder and voiding symptoms. To date,there are no effective treatments for IC. We investigated whether filteredadipose-derived stem cell lysate (FADSCL) alleviates IC in a rat model ofHCl-induced cystitis.

Materials and methods Rat adipose-derived stem cells were collectedfrom the subcutaneous fat of rats, and FADSCL (lysate of 1×107 cells/PBS) wasprepared. Female Fisher 344 rats, aged eight weeks, were divided into thesham+PBS (sham, n=10), HCl+PBS (HCl, n=12), and HCl+FADSCL (FADSCL, n=9) groups.Saline or 0.1 M HCl (250μl/body) was intravesically administered from atransurethral catheter. Bladder function was investigated after one week bycystometrography. A sample of bladder tissue was used for RNA-seq analysis.

Results The intercontraction intervals (ICIs) in the HCl group weresignificantly shorter than those in the sham group (P<0.01), while the ICIsof the FADSCL group were significantly longer than those of the HCl group(P<0.01). There was no significant difference between the groups with respectto the maximum voiding pressure and bladder weight. RNA-Seq analysis revealed anincrease in immune and inflammatory signaling in the HCl group compared to thatin the sham group, and these signals were suppressed in the FADSCL group.

Conclusion FADSCL might alleviate IC via anti-inflammatory actionsand suppress the immune response. Thus, FADSCL could serve as a noveltherapeutic agent for IC.

G-Protein coupled receptors (GPCRs) form the largest family of cell surface receptor proteins and play key roles in eukaryotic cells, including in neuron. Recently, advance of research about GPCR led to the idea that some GPCRs form complex with other GPCRs to constitute a hetero-oligomer and control their function. Such interaction of GPCRs is thought to be involved in various physiological and pathological phenomena of cells. In this report, we focused on several GPCRs expressed in neuron and examined the possibility of their hetero-oligomer formation and functional interaction. We evaluated hetero-oligomeric complex formation of GPCRs by biochemical assay and microscopic imaging. Moreover, We analyzed mutual modulation of their GPCR signalings by live cell imaging and second messenger quantification assay. Our findings give new respects to the mechanism of regulation of GPCR signaling related to neuronal function.

The hippocampus processes multimodal information associated with spatial navigation, including place, trajectory, and speed. However, how such information is distributed to multiple downstream areas remains poorly understood. We investigated this issue by identifying axonal projections using multisite optogenetics during large-scale extracellular recordings from the rat subiculum, the major hippocampal output structure. Subicular neurons demonstrated a noise-resistant representation of place, speed, and trajectory, which was as accurate as or even more accurate than that of hippocampal CA1 neurons. Speed and trajectory information was most prominently sent to the retrosplenial cortex and nucleus accumbens, respectively. Place information was distributed uniformly to the retrosplenial cortex, nucleus accumbens, anteroventral thalamus, and medial mammillary body. Information transmission by projection neurons was tightly controlled by theta oscillations and sharp-wave/ripples in a target region-specific manner. In conclusion, the dorsal subiculum robustly routes diverse navigation-associated information to downstream areas.

Integration of spatial and event information in episodic memory is thought to be supported by hippocampus. However, it is unknown how an event such as receipt of reward is represented together with spatial information at the cellular level. To address this issue, we performed longitudinal calcium imaging in hippocampal CA1 of mice performing a spatial delayed reward task in virtual reality. In this task, reward was given when mice stayed for 2 sec at one of the three different colored zones in a virtual linear track. After learning, about 13% of cells demonstrated time-locked activity while the mice waited for reward, and the proportion was correlated with the task performance. When the delay before the reward was omitted, the onset of activity shifted accordingly. In addition, 1.4% of cells continued to respond to the previous reward zone after reward delivery was moved to a new zone. Interestingly, a subset of cells that showed time-locked activity participated in multiple synchronous activity with place cells during the stationary, and the occurrence of such coactivity persisted over multiple days. These results suggest that formation and persistence of neuronal assemblies that involve cells encoding event timing and place cells may be implicated in a hippocampal memory process that associates events with space.

Post-traumatic stress disorder (PTSD) is a psychiatric disorder associated with memories of traumatic experiences. In experimental animals, fear conditioning task is the most widely used as a model of PTSD. Previous studies have suggested a key role for the cannabinoid CB₁ receptors in the regulation of conditioned fear memory. Here, we investigated that the roles of endocannabinoid in fear memory on fear conditioning task, using the inhibitors of endocannabinoid hydrolysis. URB597, an anandamide hydrolysis inhibitor, significantly suppressed the expression rate of freezing behavior in both cue-elicited and contextual fear conditioning test in ICR mice. In contrast, JZL184, a 2-arachidonoylglycerol (2-AG) hydrolysis inhibitor, significantly increased the expression rate of freezing behavior. JZL195, a dual anandamide/2-AG hydrolysis inhibitor, also increased the expression rate of freezing behavior. Furthermore, we investigated the effect of URB597, JZL184 and JZL195 on anxiety-like behaviors with the elevated-plus maze test immediately after the fear conditioning tests. None of the above three inhibitors changed the time spent in open arms and the number of crossings. These findings suggest that the endocannabinoids play bidirectional roles in retrieval of fear memory. Namely, fear memory could be regulated by endocannabinoid, in particular, suppressively by anandamide but facilitatory by 2-AG.

Many studies report that the electroconvulsive seizure (ECS) administered immediately after the training (fear conditioning) impairs the memory formation. In contrast, there are only few studies asking whether or not the ECS before the training influence the learning. Here, we trained mice by fear conditioning and investigated which timing of the ECS abrogates the memory formation. Contextual and auditory memories were tested 1 h, 24 h, and 8 d after the conditioning to assess the formation of short- (STM) and long-term memories (LTM). The ECS 2 h before the conditioning caused a decrease in contextual-freezing, but did not affect the auditory-freezing, when the memory was tested 24 h after the conditioning. However, the ECS 2 h before the conditioning did not decrease in either contextual- or auditory-freezing, when the memory was tested 1 h after the conditioning. In addition, the ECS 2 h before the conditioning did not affect the contextual- or auditory-freezing, when the memory was tested 8 days after the conditioning. The data suggest that the ECS 2 h before the conditioning inhibits the transition from the contextual STM to the LTM one. As the ECS strongly induces various immediate-early genes, some of the gene products might be involved in the abrogation of the transition of the STM to the LTM.

To evaluate perseverative or compulsive properties of genetic mice models of various neurological and psychiatric disorders, a reversal task has been used. However, because the conventional reversal tasks are limited to two options, it is difficult to distinguish between impairments in explorative propensity and impairments in the extinction of learned behaviors. Therefore, we developed a five-choice exploratory operant task - the five-arm bandit task (5-ABT) - to assess behavioral flexibility. The task consists of Phase 1 in which five options are equally rewarded with a constant probability, Phase 2 in which only one option is rewarded, and Phase 3 in which the reward pattern of Phase 2 is reversed. Using this task, we analyzed the behavior of Big Potassium Channel Knockout (BK KO) mice. The choice entropies in Phase 3 between BK KO mice and its wild type littermates were significantly different, while the entropies in Phase 1 were not different. This suggests that BK KO mice are impaired with respect to extinction of learned options or learning from mistakes rather than exploration. To quantify the ability to learn from rewarded and unrewarded experiences, we also estimated the positive and negative learning rate parameters by fitting behavioral data to a Q-learning model with differential learning rate. The negative learning rates of BK KO mice was significantly reduced compared to the wild type, indicating that their ability to learn from mistakes was impaired. This method enables the examination of candidate murine models for various neurological and psychiatric disorders, including autism and addiction, and the subsequent evaluation of pharmacological effects.

Age-related declines in physical performance (physical frailty) contribute to an increased risk for adverse events in elderly people. Ninjin’yoeito (NYT), a traditional Japanese medicine, has been used to improve physical decline in convalescent patients, symptoms of fatigue, anorexia, and anemia. In this study, we evaluated whether treatment with NYT has beneficial effects on the behavioral alterations in aged C57BL/6J mice.

We used 7-month-old male C57BL/6J mice and 20-month-old male C57BL/6J mice as the young and aged mice, respectively. Aged mice were divided into the following three groups: the control group fed a CRF-6 diet and the treatment groups fed a CRF-6 diet containing either 1% or 3% NYT extract (purchased from TSUMURA &amp; Co.). Young mice were also fed a CRF-6 diet. After 10–11 weeks of treatment, we performed behavioral assays.

We found that motor coordination (rotarod test), forelimb grip strength (grip strength test), and self-care motivation (sucrose splash test) declined in the aged mice in the control group as compared with young mice; however, these decrements were alleviated with NYT treatment.

These results demonstrate the effects of NYT in aged mice, implying that NYT has the potential to be a useful agent for improving age-related behavioral declines.

Temporal lobe epilepsy with distributed hippocampal seizure foci is oftenintractable and its secondary generalization might lead to sudden death. Earlytermination through spatially extensive hippocampal intervention is not feasibledirectly, due to the large size and irregular shape of the hippocampus. Incontrast, the medial septum (MS) is a promising target to govern hippocampaloscillations through its divergent connections to both hippocampi. Combiningthis ‘proxy intervention’ concept and precisely timed stimulation, we reporthere that closed-loop MS electrical stimulation can quickly terminateintrahippocampal seizures and suppress secondary generalization in a ratkindling model. Precise stimulus timing governed by internal seizure rhythms wasessential. Cell-type-specific stimulation revealed that the precisely timedactivation of MS GABAergic neurons underlaid the effects. Our concept oftime-targeted proxy stimulation for intervening pathological oscillations can beextrapolated to other neurological and psychiatric disorders, and has potentialfor clinical translation.

Micro-electrode array (MEA) assay using in vitro human iPSC-derived neurons is expected as one of the assessments for predicting seizure liability of drugs. However, it is necessary to approach the in vitro to in vivo extrapolation (IVIVE). As one approach to IVIVE, an assessment method using a human brain organoid that mimics the three-dimensional structure is considered to be effective. In this study, we attempted to detect the response to convulsants by MEA measurement using human cerebral organoids. We detected the responses to convulsants in cerebral organoids and found that features appear in low frequency components of MEA data. Wavelet analysis revealed that the frequency intensity from the θ wave to the β wave component significantly increased in a dose-dependent manner. This is a result that enables comparison with in vivo brain waves. This study demonstrated that MEA measurement using human brain organoids may be a method that can approach in vitro to in vivo extrapolation in prediction of seizure liability of drugs.

Most of hearing loss (HL) is caused by problems in the cochlea; however, some are from problems in the cochlear nerve linking hair cells to the brain. The anatomical structure “glial dome” demarcates the peripheral and central segment of the cochlear nerve at the cochlear modiolus: the former is myelinated by Schwann cells and located at the Rosenthal’s canal (RC) and osseous spiral lamina (OSL). Muscular dystrophies (MDs) are characterized by progressive degeneration of skeletal muscle. MD-dystroglycanopathy (MD-DG) is caused by aberrant glycosylation of α-dystroglycan, and accompanied with various non-muscular symptoms. Since no comprehensive study regarding HL in MD-DG has accomplished, we investigated two mouse models of MD-DG, LARGE-deficient and POMGnT1-KO mice. MD-DG mice showed nonprogressive HL with delayed wave I latency in auditory brainstem response (ABR). Glycosylated α-dystroglycan is rich at the RC and OSL in controls, and decreased in MD-DG cochleae. Additionally, abnormal myelination of the peripheral cochlear nerve was observed at the RC and OSL by immunostaining, immunoblotting, electronic microscopy. Furthermore, Fukuyama congenital MD, a type of MD-DG, patients showed delayed wave I latency in ABR. Thus, HL of MD-DG is caused by impaired Schwann cell-mediated myelination at the peripheral cochlear nerve.

Glaucoma is one of the most common causes of blindness in Japan. In the retina of the patients of glaucoma, retinal ganglion cells (RGC) are selectively degenerated, and glutamate-induced toxicity may be involved in the underlying mechanisms. Previously, RGC-5 was proposed to be a cell line derived from rat retinal ganglion cells and used as a useful model for the assay for glutamate-induced RGC toxicity. However, resent studies have revealed that RGC-5 is not rat RGC, but murine photoreceptor cells. Recently, QNR/D cells, a cell line derived from quail embryotic neruroretinas, were reported and the cell line can be obtained from ATCC. In the present study, we tried to establish an assay system for protective agents against glutamate-induced toxicity using QNR/D cells. QNR/D cells were cultured in DMEM supplemented with 10% FBS in 5% CO2/95% O2 at 39 degree Celsius. Cell survival was determined by WST-8 assay. Under the presence of L-buthionine-(S,R)-sulfoximine (2 mM), which depletes cellular glutathione levels, glutamate (4-25 mM) concentration-dependently induced cell death. This cell death was reduced by vitamin E (100 µM), but not by MK-801, an N-methyl-D-aspartic acid (NMDA) receptor antagonist, nor L-nitro-L-arginine methyl ester, an NO synthase inhibitor. NMDA did not induce cell death. These results suggest that protective agents not against excitotoxicity, but against oxidative glutamate toxicity can be assayed using the present experimental system.

Glaucoma is the leading cause of blindness worldwide and the blindness is caused by the degeneration of retinal ganglion cells (RGCs). Although an elevated intraocular pressure (IOP) is widely accepted to be the major risk factor, many patients, especially Japanese, show normal IOP levels (i.e. normal-tension glaucoma, NTG). We have recently discovered a novel NTG model mouse in which astrocytes lack the gene encoding ATP-binding cassette transporter A1 (ABCA1). The NTG mice at a young age (3 months old) showed no RGC damages but showed significant damages of RGCs and visual impairment at middle-age (12 months old). We then investigated molecular mechanisms triggering NTG and focused on optic nerve head (ONH). Excavation of ONH is a common anatomical feature of both hypertensive glaucoma and NTG. We performed RNA-seq and found that a young ONH showed the largest number of gene expression changes. Immunohistochemical analysis showed that ONH astrocytes at a young age already showed reactive gliosis with some tissue remodeling at the periphery. A profile for astrocyte marker genes showed no specific shifts to the neurotoxic phenotype. Gene ontology analysis revealed that remodeling of the extracellular matrix (ECM) was the most relevant biological event in the young ONH. ECM such as collagen IV was highly expressed in the ONH astrocytes. Taken together, our data demonstrated that astrocytes become reactive in the ONH at a young age when RGCs showed no damages. The reactive astrocytes triggered ECM remodeling which would cause excavation of ONH and RGC damages.

Glaucoma is second leading cause of blindness worldwide which is characterized by progressive degeneration of retinal ganglion cells (RGCs).  Although an elevated intraocular pressure (IOP) is major risk factor, the mechanism for IOP regulation has not fully understood. Here we report that the P2Y₁ receptor (P2Y₁R) is essential for IOP reduction and its dysfunction causes ocular hypertensive glaucoma-like phenotypes. P2Y₁R activation by MRS2365, a selective agonist for P2Y₁R, significantly reduced IOP in wild-type (WT) mice but not in P2Y₁KO mice. P2Y₁R was dominantly expressed in the ciliary body and the angle tissue including trabecular meshwork and Schlemm's canal, essential for aqueous humor (AH) production and draining, respectively. Supporting this observation, P2Y₁R activation suppressed production and enhanced draining of AH, respectively. We also found that aquaporin 4 (AQP4) is downstream target of P2Y₁R. AQP4 was expressed in the ciliary body, which was well co-localized with P2Y₁R-positive signals. The findings that an AQP4 blocker significantly suppressed AH production, which was not reduced further by MRS2365 suggest that P2Y₁R and AQP4 should share the same pathway for the AH reduction. P2Y₁KO mice showed chronic ocular hypertension. P2Y₁KO mice at 12 months old showed significantly lower RGC number, thinner retina, optic nerve atrophy and impaired visual function. Taken together, our results demonstrated that P2Y₁ receptor activation reduces IOP and P2Y₁KO mice show hypertensive glaucoma-like phenotypes.

Recent evidence suggests that manipulation of astrocytic GqPCR can alter animal behavior related to learning and memory through modulation of neuronal functions. It has been shown that impairment of GqPCR-mediated Ca²⁺ signaling in astrocytes and/or gliotransmitter release causes depression-like behaviors in mice. However, it is not clear whether astrocytic GqPCR signaling is a target for driving behavior related to stress and motivation. We focused on the ventral hippocampus (vHIP), which contributes to stress and depression, and investigated the effect of chemogenetic manipulation of astrocytic GpPCR signaling in animal behavior related to stress. We expressed hM3Dq, a Gq-DREADD, selectively in astrocytes in the vHIP by injecting AAV bilaterally. Activation of hM3Dq caused robust Ca²⁺ elevation in astrocytes and modulated synaptic transmission. Astrocytic GpPCR activation did not alter place preference assessed by open filed test, suggesting no increase in fear-related behavior. Chronic but not acute administration of hM3Dq agonist decreased immobility time in the tail suspension test. These results suggest that chronic activation of GqPCR signaling in vHIP astrocytes should enhance active coping in response to stress and chemogenetic manipulation of astrocytic Ca²⁺ signaling in the vHIP may enhance resilience to stress.

Abnormalities of astrocytic roles are implicated in neuronal hyperexcitablity of neurological diseases. In many pathological conditions, astrocytes enhance Ca²⁺ signals by increasing Gq-protein coupled receptors (GqPCR) including P2Y1 receptor. To understand the role of the GqPCR augmentation, we have investigated astrocyte specific overexpression mice model of P2Y1 receptor (P2Y1OE). Performing simultaneous imaging of neurons and astrocytes in the hippocampal CA1 region, we found that electrical stimulation of the Schaffer collateral resulted in fast Ca²⁺ rise in dendrites of neurons followed by slow-onset Ca²⁺ rise in astrocytes. Fast Ca²⁺ rise in dendrites was augmented in P2Y1OE and mediated by ionotropic glutamate receptors. Also, fast dendritic Ca ²⁺ was reduced by inhibition of slow-onset Ca²⁺ rise in astrocytes. Pharmacological data suggest augmented dendritic Ca²⁺ is due to glutamate release. Extracellular glutamate imaging data suggest that the glutamate release is derived from neurons but not astrocytes. Transcriptome analysis of isolated astrocytes from P2Y1OE revealed a novel candidate molecule X as an astrocyte-derived excitatory signal, which could underlie astrocyte P2Y1-mediated neuronal excitation through enhancement of excitatory synaptic transmission.

Information processing in the brain is performed not only by neurons but also by glial cells. Specifically, a major glial cell type, the astrocyte, is in charge of forming and maturing synapses to establish sustainable synaptic transmission in the brain. Generally, higher animals have larger brains. There is a possibility that the number of astrocytes determines the intricate brain function in which a higher animal’s brain has a higher density of astrocytes. This study took advantage of a primary co-culture system using a single autaptic hippocampal neuron with dot-patterned cortical astrocytes. This preparation enables the proper and systematic counting of astrocytes surrounding a single neuron. As a result, a hippocampal neuron with a higher density of astrocytes showed more excellent excitatory synaptic transmission than that of neurons with a lower density of astrocytes. This result was accompanied by a significant increase in the pool of readily releasable synaptic vesicles. The number of morphologically identified glutamatergic synapses was comparable, but the percentage of functional ones was increased, indicating a lower ratio of presynaptically silent synapses. Taken together, the higher astrocytic density enhanced excitatory synaptic transmission by increasing the fraction of functional synapses through presynaptic un-silencing.

Astrocytes are glial cells with numerous processes through which they contact neurons to control functions in the central nervous system (CNS). Astrocytic processes are morphologically heterogeneous throughout the CNS regions and exhibit neuronal activity-induced plasticity. We previously reported that the process formation by cultured spinal cord astrocytes is bidirectionally regulated by β- and α₂-adrenoceptors (ARs). Here, we examined the role of α₁-ARs in process formation and adrenergic regulation of cortical astrocyte processes. We evaluated morphology of cultured astrocytes by phalloidin-cytoskeletal staining.

Noradrenaline (NA) and isoproterenol (β-agonist) induced process formation, which was more potent in cortical astrocytes than in spinal cord astrocytes. Prazosin and atipamezole (α₁- and α₂-antagonist) enhanced NA-induced process formation in spinal cord astrocytes but not in cortical astrocytes. Dexmedetomidine (α₂-agonist), but not phenylephrine (α₁-agonist), inhibited the process formation by both astrocytes. The expression of AR mRNAs was different between both astrocytes, especially less expression of α₁-ARs in cortical astrocytes.

 Adrenergic regulation of astrocyte processes likely depends on the balance of α- and β- ARs, which may be related to astrocytic morphological heterogeneity and functions in the CNS.

Hydrogen sulfide (H₂S), which is produced by astrocytes in the central nervous system, inhibits the mitochondrial electron transport chain (ETC). We have previously shown that H₂S releases Ca²⁺ from the endoplasmic reticulum (ER) in spinal cord astrocytes. Here, we examined the relationship between H₂S-induced metabolic changes and Ca²⁺ response.

Intracellular Ca²⁺ concentration ([Ca²⁺]_i) in cultured rat spinal cord astrocytes was measured using Fura 2-AM. Na₂S was used as a H₂S donor. The extracellular lactate and intracellular ATP were measured by enzymatic reaction using lactate dehydrogenase and luciferase, respectively.

Na₂S (150 µM) increased [Ca²⁺]_i, which was inhibited by rotenone, an ETC inhibitor, and FCCP, an uncoupler of oxidative phosphorylation. Na₂S also increased extracellular lactate, and decreased intracellular ATP content when glycolysis was inhibited by iodoacetic acid. The increase in both Ca²⁺ and extracellular lactate by Na₂S were inhibited by emetine, an inhibitor of translocon complex, which mediates Ca²⁺ leak from the ER.

In conclusion, inhibition of the mitochondrial ETC by H₂S induces Ca²⁺ release from the ER and lactate production in spinal cord astrocytes. H₂S may facilitate the supply of lactate from astrocytes to neurons as an energy substrate.

Histidine-rich glycoprotein (HRG) is an anti-inflammatory factor that controls the progression of systemic inflammatory pathology. In more severe inflammatory condition such as sepsis syndrome, the reduced level of plasma HRG is exacerbating the mortality by inducing to the neutrophil hyperactivity, the promotion of blood coagulation cascade, the vascular endothelial dysfunction, and so on. Although HRG is mainly produced from liver and present at high level of 100 ug/mL in plasma, the physiological functions of plasma HRG on healthy conditions have not been clarified yet. Recently, it was reported that an excess reduction of plasma HRG level in human pregnant was correlated with the hypertensive disorders of pregnancy (HDP) seriousness. In this study, we examined the involvement of plasma HRG on the gestational period using C57BL/6 mice and HRG gene-deficient (HRG KO) mice. Plasma HRG level was decreased during the healthy gestational period in C57BL/6 mice. In addition, HRG reduction was restored at post-partum and did not depend on the HRG gene expression in liver. Hysterectomy to the gestational C57BL/6 mice also restored the plasma HRG level up to the normal level. When Human plasma-purified HRG was injected to C57BL/6 mice on nulliparity or gestation, biological half-time of the injected HRG was short in the gestational mice compared with the nulliparous mice. Moreover, HRG KO mice showed hypertension by the gestation, whereas rise in blood pressure did not observed in the healthy gestational C57BL/6 mice. These results suggest that HRG may have a physiological function in gestational period. Pregnant HRG KO mice may be a HDP-like pathological model.