Gangliosides are abundant sialic acids containing glycosphingolipids in neural tissues. We reported that intraplantar injection of ganglioside GT1b caused mechanical allodynia and Arthrobacter ureafaciens sialidase that degrades sialyl conjugates suppressed inflammatory pain. Moreover, F-11 cells (a cell line derived from the dorsal root ganglion neuron) treated with sialidase showed shortened neurites. Several studies reported that the number of nerve fibers in the epidermis is increased during skin inflammation. Thus, we investigated whether sialidase affected the epidermal nerve fiber innervation.

Sialidase injection into the inflamed hind paw of mice one day after injection of complete Freund's adjuvant decreased the length of epidermal nerve fibers. However, it is unclear that the localization of gangliosides in the nerve fibers in the skin tissues. Anti-GT1b antibody stained nerve fibers in the dermis near the epidermal-dermal junction during inflammation, whereas nerve fibers in the naïve hind paw showed poor staining with this antibody. These results indicate that skin inflammation leads to the accumulation of the gangliosides in the nerve fibers, which contributes to nerve fiber elongation.

Olanzapine is an antipsychotic drug. Weight gain is a typical side effect of olanzapine, but its mechanism is unknown. Leptin is a centrally acting anti-obesity hormone, involved in attenuating obesity. It has been shown that leptin resistance is involved in diet-induced obesity. In the present study, we hypothesized that mechanisms of olanzapine on weight gain may involve leptin resistance.We performed leptin stimulation on SHSY-5Y-ObRb cells expressing leptin receptor and examined the effect of olanzapine on phosphorylation of STAT3, an indicator of leptin signaling. We found that in the presence of olanzapine, leptin-induced phosphorylation of STAT3 was inhibited in a dose-dependent manner. On the other hand, olanzapine did not inhibit IL-6-induced STAT3 phosphorylation. These results suggest that the inhibition of STAT3 phosphorylation may be leptin receptor specific. In order to investigate the possibility that olanzapine induces leptin resistance through the translation synthesis, we conducted the same experiment in the presence of the translation inhibitor, cycloheximide. As a result, cycloheximide had no effect on the inhibition of STAT3 phosphorylation by olanzapine. This indicates that the inhibition of leptin-induced STAT3 phosphorylation by olanzapine may not involve protein synthesis.

Pancreatic β-cells release insulin to control glycemia in an intracellular Ca²⁺-dependent manner. Spatiotemporal dynamics and the function of Ca²⁺ signaling in β-cells have been studied by experiments using in vitro and ex vivo preparations. However, in vivo study remains challenging. Unlike the case of in vitro/ex vivo preparations, β-cells in vivo are under the influence of the autonomic nervous system, hormones, and other bioactive substances. Thus, β-cell Ca²⁺ activities 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 ratiometric Ca²⁺ indicator protein, 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 provide unprecedented clues to understand the regulation mechanism of intravital insulin dynamics. Further studies in living animals using the new method are expected to help elucidate the mechanism of insulin secretion and the etiology of diabetes.

The voltage-dependent L-type calcium channel (L-VDCC) plays an essential role in regulating insulin secretion in pancreatic β-cell. Excessive calcium signaling due to metabolic stress has been reported to cause β-cell dysfunction and cell death. Therefore, the fine-tuning of calcium signaling is critical for the homeostatic regulation of insulin secretion. Dyslipidemia in type 2 diabetes is known to impair insulin secretion. However, the underlying mechanism has not been fully elucidated so far. In the present study, we focused on the relationship between calcium signaling and lipotoxicity to elucidate the molecular mechanism of insulin secretory failure. Protein expression of L-VDCC α₁ subunit Ca_V1.2 in pancreatic β-cell line MIN6 was detected by flowcytometry and western blotting. When MIN6 cells were cultured in the presence of certain fatty acids, the protein expression level of Ca_V1.2 was significantly reduced. We confirmed that there was no change in the gene expression level of Ca_V1.2. The fatty acid-induced reduction of Ca_V1.2 protein was blunted by MG132, a proteasome inhibitor, suggesting that fatty acid promoted the degradation of Ca_V1.2. These results indicate that a novel lipotoxic mechanism in degrading L-VDCC Ca_V1.2 may be involved in dyslipidemia-induced insulin secretory failure.

Probiotics, including Lactobacillus and Bifidobacterium, is live microorganisms which, when administered in adequate amounts, confer a health benefit on the host, such as intestinal conditioning and anti-inflammatory effects. However, the intestinal protective molecules derived from probiotic bacteria has been less identified. In this study, we identified inorganic long chain polyphosphate as the intestinal protective molecules from the culture supernatant of Lactobacillus brevis SBC8803 by using HPLC and elemental analysis. Polyphosphate activated integrin β1-p38MAPK signaling pathway and its intestinal protective effect was diminished by the inhibition of epithelial integrin. Polyphosphate was recognized by integrin, incorporated to epithelial cells through endocytic pathway from apical side, and then released to apical side. Polyphosphate improved the intestinal injury in rat DSS induced colitis model and investigator initiated clinical trial targeted to ulcerative colitis patients. Therefore, probiotic derived polyphosphate will be applied for treatment of refractory intestinal bowel disease.

Kinase inhibitors are potent cancer therapeutics. However, drug resistance often develops due to activation of accessory signaling pathways. Multi-target kinase inhibitors have thus been tried although their benefit and risk remain largely elusive. In this regard, we recently discovered an alerting mechanism by which anti-cancer drugs may become cancer-promoting agents (Cell Rep. 34:108876, 2021). We found that c-Src which normally locates on the plasma membrane, massively translocates to focal adhesions upon treatment with various tyrosine kinase inhibitors. Inhibitor-binding to the Src kinase domain causes the relief of autoinhibition, and released Src SH3 and SH2 domains bind focal adhesion kinase (FAK). At this step, Src is inactive. However, the low-affinity Src inhibitors such as AEE788, whose primary targets are EGFR and HER2, could be readily washed out from the Src-FAK complex, which leads to phosphorylation of FAK by Src. This triggers activation of Erk signaling cascade by recruiting Grb2 to focal adhesions. Furthermore, when MCF7 cells harbor drug-resistant mutations in SRC gene, even high-affinity inhibitors such as dasatinib and bosutinib induce the activation of the Src-FAK-Grb2-Erk signaling, paradoxically promoting cancer cell proliferation. We will discuss the implications of allosteric effects of ATP-competitive inhibitors and the paradoxical activation of cancer promoting signaling by kinase inhibitors.

[Introduction]

Fibrosis is the final common pathway of chronic kidney disease, leading to end-stage renal failure. In our previous data, the transcript expression of leupaxin, a member of paxillin protein family, was upregulated in the fibrotic kidneys, analyzed by microarray. However, the role of leupaxin in kidney fibrosis is unclear. The aim of this study is to assess the function of leupaxin in kidney fibroblasts which play critical roles in kidney fibrosis.

[Methods and Results]

Kidney fibrosis model was generated by ligating the unilateral ureter of C57Bl/6J mice (UUO). Quantitative PCR analysis showed that leupaxin mRNA expression was increased 1, 3 and 7 days after UUO. Moreover, immunohistochemical staining revealed that leupaxin was expressed in the fibrotic area 7 days after UUO. Next, we explored the function of leupaxin in fibroblasts using cultured rat renal fibroblasts NRK49F cells. Immunostaining and western blotting demonstrated that leupaxin localized in the cell nuclei. Finally, The knock-down of leupaxin by lentiviral shRNA resulted in the reduced proliferative capacity of NRK49F cells.

[Conclusion]

Leupaxin regulates the proliferation of kidney fibroblasts as a nuclear protein. The suppression of leupaxin could be a therapeutic approach for kidney fibrosis.

Acute kidney injury (AKI) is a pathological condition in which renal function rapidly decreases due to ischemia/reperfusion or drugs, and can lead to the end-stage renal disease. However, there are no approved effective therapeutics for AKI. Recent studies have suggested that inflammation and oxidative stress are the primary causes of AKI. We previously reported the potential anti-inflammatory and antioxidant activities of Stachybotrys microspora triprenyl phenol-7 (SMTP-7). The aim of the present study was to evaluate the efficacy of SMTP-7 in AKI model mice. AKI was induced in mice by ischemia of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after the removal of right kidney. The efficacy of SMTP-7 was determined by measuring the renal function using urine and serum samples and morphological assessment. For deciphering the mechanism of action of SMTP-7, inflammatory cytokines and oxidative stress in kidney were detected. SMTP-7 (0.01, 0.1, 1, 10 mg/kg) dose-dependently improved the renal function. In addition, it improved the damage to renal tubules and exhibited anti-inflammatory and antioxidant activities in the kidney of AKI mice. In conclusion, based on the data obtained in the present study, these results indicate the potential of SMTP-7 as a medicinal compound for the treatment of AKI.

6R-L-erythro-5,6,7,8-Tetrahydrobiopterin (BH4) is an essential cofactor for aromatic L-amino acid hydroxylases including tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine-biosynthesis, and for all types of nitric oxide synthases (NOS). Sepiapterin reductase (SPR) catalyzes the third step of BH4 biosynthesis. SPR gene-disrupted (Spr^-/-) mice (OYC32, Lexicon Pharmaceuticals Inc.) exhibit a dystonic posture, low body weight, hyperphenylalaninemia, and unstable cardiovascular control with endothelial dysfunction. Recently, we found that Spr^-/- mice suffered from severe priapism (persistent erection) at a high incidence. This priapism was relieved by repeated administration of BH4. We hypothesized that the priapism of Spr^-/- mice was primarily caused by sympathetic nerve failure due to cofactor depletion and the loss of TH protein, which caused vasodilation and relaxation of the corpus cavernosum. Its disinhibition of neuronal NOS (nNOS)-containing neurons would cause overproduction of NO and further exacerbated the priapism. To verify the possibility, we analyzed biopterin and norepinephrine content, TH protein amount and nitric oxide metabolites in the penile tissue of Spr^-/- mice with and without BH4-supplementation.

Objectives: The anti-cancer drug doxorubicin (DOX) can induce erectile dysfunction (ED) and decrease testosterone levels. We investigated the effect of testosterone replacement therapy (TRT) on ED by DOX.

Methods: Twelve-week-old male Wistar-ST rats were untreated (Control) or administered DOX (3 mg/kg i.v. weekly for 4 weeks) in DOX and DOX+T groups. Testosterone undecanoate (25 mg/kg s.c.) was administered in the DOX+T group. Erectile and endothelial functions were measured using intracavernosal pressure (ICP) and isometric tension. Expressions of inflammation and oxidative stress markers (NADPH oxidase-1, IL-6) were assessed via quantitative PCR.

Results: ICP/MAP ratio in the DOX group (0.32 ± 0.03) was significantly decreased compared to the Control group (0.72 ± 0.05). The ratio was significantly increased in the DOX+T group (0.61 ± 0.07). Acetylcholine reactivity was significantly lower in DOX group than in Control group (35.0 ± 5.1% vs 77.6 ± 7.9%) and was higher in DOX+T group (69.8 ± 8.5%). NADPH oxidase-1 and IL-6 mRNA expressions were higher in DOX group and lower in DOX+T group.

Conclusion: TRT in rats may improve ED induced by DOX. This is likely because TRT improved vascular endothelial function by suppressing inflammatory response. TRT may be a treatment option for symptoms of androgen deficiency following anticancer drug therapy.

Introduction: Fibrin(ogen) plays an important role in various physiological processes and is also critical for maintaining fetal-maternal attachment during pregnancy. Fibrin has been used to increase implantation rates in human-assisted reproductive technology; however, its utility in vitro has not yet been demonstrated. 

Results: Fibrin did not affect the developmental speed or morphology of mouse blastocysts, and a large fibrin-degrading region was observed in the attachment stage. On the other hand, fibrin significantly suppressed the spreading and outgrowth of trophoblast in human blastocysts, and trophoblast grew after the appearance of small fibrin-degrading regions. uPA was identified as a fibrinolytic factor in the supernatant, and uPA activity was significantly weaker in human blastocysts than in mouse blastocysts. The inhibition of uPA significantly reduced the outgrowth of trophoblast in mouse and human blastocysts. Human blastocysts expressed PLAU (uPA), PLAUR (uPAR), SERPINE1 (PAI-1), and SERPINB2 (PAI-2), whereas mouse blastocysts were limited to Plau, Plaur, and Serpine1. In a subsequent experiment on human blastocysts, the addition of exogenous uPA and the PAI-1 inhibitor promoted trophoblast growth in the presence of fibrin, as did the addition of FDPs.

Discussion: The present results suggest that the distinct features of trophoblast spreading and outgrowth in human blastocysts observed in the presence of fibrin are regulated by a phenotypic conversion induced by contact with fibrin and FDPs. Mouse embryos did not reproduce the human phenomenon, indicating that the present results are unique to humans.

Cancer chemotherapy often induces taste disturbance that reduces quality of life of the cancer patients. However, the mechanisms underlying chemotherapy-induced taste disturbance are poorly understood. It is known that anti-cancer agents that cause taste disturbance, such as platinum agents, also induces peripheral neuropathy that based on neurotoxicity. In this study, we investigated the alteration of the gustatory nerve and taste receptors in the cisplatin-induced taste disturbance model rats. We made taste disturbance model by repeated administrations of cisplatin (2 mg/kg, i.p.) once a week for 8 weeks. The taste sensitivity to brief-access test was significantly decreased in cisplatin-administered rats on 5 and 8 weeks, compared to control group. Consistently, in electron microscopic analysis, the axonal degeneration in the gustatory nerve was observed on 5 and 8 weeks. However, the expression levelof sweet taste receptor T1R2mRNA in the taste bud was decreased only on 8 weeks, and that of bitter taste receptor T2R107 mRNAwas not changed. These data show that alteration of taste sensitivity was correlated with axonal degeneration in gustatory nerve, rather than that of taste receptor level. These results suggest that the taste disturbanceinduced by cisplatinis related to the gustatory nerve degeneration.

Background) Genomic DNA, which contains all of the genetic information, is damaged by a variety of endogenous and environmental factors such as genotoxic chemicals, ionizing radiation and UV light. Consequently, the DNA repair process is constantly active as it responds to damage in the DNA structure. Not only cardiotoxicity of anticancer drug treatment but also ischemic heart disease and heart failure associated with overloaded pressure interfere with DNA damage response and DNA repair regulation in cardiomyocytes. We have reported that anticancer drug treatment causes a transient decrease in plasma brain natriuretic peptide (BNP) levels, but the mechanism of this decrease is unknown. In the present study, to clarify this molecular mechanism, we analyzed mouse hearts treated with anticancer drugs. Methods) C57BL/6 mice were treated with cisplatin (16 mg/kg, ip), and then BNP gene expression and DNA methylation in the heart were examined. Results) In cisplatin-treated mice, there was a transient decrease in BNP gene expression and an increase in DNA methylation of the BNP promoter region. Furthermore, DNA methyltransferase type 1 (DNMT1) was found to affect this DNA methylation. Conclusions) In cisplatin-treated mice, cardiac BNP transcription was repressed by DNA methylation. DNMT1 was suggested to be involved in this DNA methylation modification.

Multicellular tumor three-dimensional spheroid models are spherical self-assembled aggregates of cancer cells. They are a valuable tool for studying the tumor microenvironment in solid tumors and are also important for investigating the characteristics of cancer stem cells such as chemoresistance. Spheroid formation of human prostate cancer (PC) LNCaP cells with ultra-low attachment surface cultureware induced the up-regulation of cancer stem cell markers, and decreased the protein degradation of the Ca²⁺-activated K⁺ channel K_Ca1.1 by down-regulating the E3 ubiquitin ligase, FBXW7. K_Ca1.1 activator-induced hyperpolarizing responses were larger in isolated cells from LNCaP spheroids. The pharmacological inhibition of K_Ca1.1 overcame the resistance of LNCaP spheroids to antiandrogens and doxorubicin (DOX). The protein expression of androgen receptors (AR) was significantly decreased by LNCaP spheroid formation, and reversed by K_Ca1.1 inhibition. The inhibition of the E3 ubiquitin ligase, MDM2, which may be related to AR protein degradation in PC stem cells, revealed that MDM2 was responsible for the acquisition of antiandrogen resistance in LNCaP spheroids, which was overcome by K_Ca1.1 inhibition. Additionally, a member of the multidrug resistance-associated protein subfamily of ABC transporters, MRP5 was responsible for the acquisition of DOX resistance in LNCaP spheroids, which was also overcome by K_Ca1.1 inhibition. Collectively, the present results suggest the potential of K_Ca1.1 in LNCaP spheroids, which mimic PC stem cells, as a therapeutic target for overcoming antiandrogen- and DOX-resistance in PC cells. These suggest that the potential of K_Ca1.1 inhibitors as an effective therapeutic intervention in combination with antiandrogens.

Protein-protein interactions (PPIs) play critical roles in the regulation of biological processes and the development of diseases. Our group found that THG-1 (TSC22 homologous gene-1) localized in the basal layer of normal squamous epithelium and overexpressed in squamous cell carcinomas. THG-1 is phosphorylated by the EGF-Ras-ERK pathway which is essential for promoting tumorigenesis. We also identified several THG-1 interacting proteins. Among them, Keap1 (Kelch-like ECH-associated protein1)-THG-1 interaction plays a crucial role in tumor progression. Therefore, inhibition of Keap1-THG-1 interaction is thought to be important for tumor suppression. In this study, we established a system to quantify the Keap1-THG-1 interaction using Nano-luc system, and performed drug repositioning screening of a chemical library from Univ. Tokyo. We identified several compounds as THG-1-Keap1 interaction inhibitor. Among these, a compound suppressed pNQO-1-ARE-luc and pHO-1-luc, suggesting that it suppresses the Nrf2 transcriptional activity. These results indicated that our screening system successfully screen the inhibitors of THG-1-Keap1 interaction. Further evaluation and large scale screening are required for future drug development.

[Introduction]

Dog bladder cancer is almost a muscle-invasive type of cancer, and many have poor prognoses due to low sensitivity to chemotherapy and difficulty in complete surgical resection. However, the obvious cause and mechanism, as well as effective treatment and prevention, have not yet been identified. Three-dimensional organoid culture is a method that can reproduce three-dimensional epithelial tissue structures of various organs on culture dishes. So far, human and mouse bladder organoids have existed, but organoids from healthy dogs have not yet been established. Therefore, we conducted this study with the aim of applying them to various researches, such as understanding the development mechanism of bladder cancer in dogs.

[Methods and Results]

The established organoids showed stable growth without bacterial contamination and were observed as spherical morphology. The organoids reproduced the layered structure of the uroepithelium, expressed uroepithelial markers such as CK7 and UPKIIIA, and showed different drug sensitivity among strains. In addition, histopathological images, dependence on culture medium components, gene expression, and protein expression were different from those of canine bladder cancer organoids.

[Conclusion]

In this study, canine normal bladder organoids were produced using a minimally invasive sample collection method, and continuous culture was possible. The organoids have a structure similar to that of the bladder in vivo, suggesting that they are useful as an experimental model of the canine normal bladder.

Maternal use of valproic acid (VPA) during pregnancy is associated with an increased risk of autism spectrum disorder (ASD) in the offspring. In the pathophysiological hypothesis of ASD, excitation/inhibition (E/I) imbalance is attracted. Dysfunction of serotonergic system is also suggested to be involved in ASD. In this study, we investigated glutamatergic-serotonergic neuronal interaction in the ASD-like behavior induced by prenatal VPA exposure in mice. Prenatal VPA exposure induced not only excessive repetitive self-grooming behavior, impairments of social behavior and object recognition memory, but also increased glutamatergic signaling (CaMKII phosphorylation) and decreased serotonin contents in the prefrontal cortex. Memantine (low-affinity NMDA antagonist) suppressed both the increase of CaMKII phosphorylation and ASD-like behaviors. Activation of serotonergic signaling via 5-HT_1A receptor by fluoxetine, tandospirone (5-HT_1A receptor agonist) and optogenetics attenuated the ASD-like behaviors in prenatal VPA-exposed mice. WAY-100635 (5-HT_1A receptor antagonist) antagonized the effect of fluoxetine on the ASD-like behaviors. These results suggest that the hyper-NMDA receptor signaling and ASD-like behaviors are associated with hypo-signaling of 5-HT_1A receptor in the prenatal VPA-exposed mice.

Dopamine contributes to attention as a key neurotransmitter in the nervous system projecting to the forebrain. Numerous animal models have been generated to model attentional impairment with dopaminergic dysregulation. We previously generated mice lacking Shati, an N-acetyltransferase-8-like protein, on a C57BL/6J genetic background (Shati/Nat8l^−/−). Modulation of this gene leads to changes in behavioral phenotypes such as attentional impairment associated with hypodopaminergic neurotransmission in mice. In this study, the effect of Shati/Nat8l deficit in dopaminergic projections was investigated using an object-based attention test (OBAT). Shati/Nat8l^−/− mice showed attentional impairment in the OBAT, accompanied by reduced neuronal activity in the prefrontal cortex (PFC) and the hypodopaminergic function indicated by the reduced expression of tyrosine hydroxylase (dopaminergic marker) protein and dopamine-related genes in the ventral tegmental area (VTA). The activation of dopaminergic projections of the VTA-PFC by chemogenetic stimulation ameliorated the attentional impairment in Shati/Nat8l^−/− mice. These results confirm previous findings that Shati/Nat8l deficiency interrupts the dopaminergic system and contributes to novel evidence that Shati/Nat8l is implicated in the dopaminergic projections of the VTA-PFC, which play important roles in the regulation of attention in the OBAT.

Valproic acid (VPA) is a widely used anti-epileptic drug. If administered to pregnant women, it causes autism spectrum disorder (ASD) in their progeny, however, the molecular mechanism by which VPA causes ASD remains unknown. Since it is reported that VPA inhibits several classes of histone deacetylases (HDACs) that regulate gene expression, we hypothesized that the alteration in autism-related gene expression is involved in VPA-induced ASD pathogenesis. Utilizing neuroblastoma cell line Neuro2a, we found that VPA reduces mRNA level of Setd5 gene whose heterozygous loss-of-function mutations are identified in autism patients. To investigate whether HDACs are involved in the regulation of Setd5 expression, we then treated another HDAC inhibitor Entinostat which inhibits class I HDACs, revealing that Entinostat also reduces Setd5 mRNA. Class I HDACs is constituted by HDAC1, HDAC2, HDAC3 and HDAC8. To determine which HDAC regulates Setd5 expression, we then individually deleted these genes and identified HDAC3 that positively regulate Setd5 expression, which is contrary to the well-established function of HDAC3 that represses gene expression. Therefore, we speculated that HDAC3 indirectly controls Setd5 expression. Consistently, inhibition of de novo protein synthesis rescued VPA-induced downregulation of Setd5 mRNA. Taken together, our data suggest that VPA represses Setd5 expression that is mediated by HDAC3-regulated gene, which at least in part contributes to ASD pathogenesis.

Whereas virgin male mice show biting attack toward pup, mice who experienced mating and delivery of partner female show paternal behavior. The medial preoptic area (MPOA) is one of the most important area to control the behavioral pattern, attack or paternal. Our previous data suggest that the GABAergic synaptic inputs into the MPOA, presumably from medial amygdala (Me), were suppressed in the male with experience of mating and cohousing with pregnant female (father mice in gestation experience; FGE mice). However, it is not clear which components of "experiences with female" have effects to initiate plastic changes at the synaptic inputs into the MPOA. To address the effects of pregnant female exposure, virgin male stayed with pregnant female for 3 days. Then, we prepared brain slices derived from these mice and performed whole-cell patch-clamp recording from MPOA neurons. The amplitude of electrically evoked inhibitory post-synaptic potentials (eIPSP) was not different between virgin and virgin without mating experience. Next, we addressed the possibility that plastic changes in the MPOA was initiated by mating without cohousing until late pregnancy. The male mice were cohoused with partner female for 4 days and then isolated. After partner female got late pregnant, we obtained brain slices from this male and compared eIPSP amplitude. However, the eIPSP amplitude was not different compared with virgin male mice. We concluded that both mating and cohabitation until late pregnancy are required to induce female experience -dependent changes of inhibitory synaptic input in the MPOA of male mice.

Primary cilia are immobile structures that extend from the surface of various cells and have been suggested to be involved in signal transduction from extracellular stimuli. In the nervous system, primary cilia are constructed in neurons and glial cells and have been suggested to play an important role in neurodevelopment and neuroregeneration, but the detailed molecular mechanisms remain unexplored. In this study, we investigated the role of primary cilia in the repair process of the damaged brain, by using the zebrafish brain injury-repair model. Zebrafish has a higher regenerative capacity than mammals and is a suitable animal model for observing the regeneration process of damaged brains. We also analyzed the effect of trichoplein and KCTD17 that we have previously identified as regulators of primary cilia formation. Trichoplein is localized in the basal bodies of primary cilia and works as a suppressor of primary cilia formation through activation of Aurora A kinase. KCTD17 is also involved in primary cilia formation through ubiquitination and degradation of trichoplein.  In this presentation, we demonstrate the role of primary cilia and the effect of trichoplein and KCTD17 in the repairing process after brain injury.

Osteoarthritis (OA) is the most common type of cartilage disease and results from the degradation of the extracellular matrix (ECM) in the joints. Approximately, more than 15 million people suffer from OA in Japan. OA also brings with it an extremely high economic burden due to being the most common form of joint disease.

Articular cartilage consists of chondrocytes and ECM molecules including aggrecan, type II, IX, and XI collagens, and hyaluronan (HA). Aggrecan and collagen are degraded in OA cartilage due to the activity of matrix metalloproteinases and aggrecanases.

The accumulation of advanced glycation end products (AGEs) is widely considered as a part of normal aging, especially in long-lived tissues. The present study was aimed to investigate the relationship between the long-term treatment response, of AGE3 effects on chondrocytes. 

Method

The chondrocyte-like cell line OUMS-27 cells were stimulated with certain doses of AGE3 (1,5,10,20,50,100,200 ug/ml). Total RNA and protein were collected from cultured OUMS-27 cells after 1,2,4,8,16 week. The levels of mRNA and protein expression for aggrecan, MMPs and Collagen were analyzed with quantitative RT-PCR and Western Blot analysis, respectively.

Result

When human cartilage-like OUMS-27 cells were cultured with AGE3 for a long period of time (8w), the production capacity of type II collagen and aggrecan was significantly reduced. Particularly at low concentrations of AGE3 (10 ug/ml), the expression level of collagen was decreased and did not change at 50 ug/ml or higher concentration. Furthermore, no statistically significant difference in all concentrations for proteases or inflammatory cytokines were obtained.

Conclusion

Prolonged exposure of chondrocytes with AGE3 reduced the expression levels of type II collagen and aggrecan, which are essential for cartilage homeostasis. It is planning to investigate the molecular mechanism of this suppression further.

[Background] To achieve the disease modeling of skeletal dysplasia, human induced pluripotent stem cells (iPSCs) will be useful but the induction method for limb bud mesenchymal (LBM) cells, which can give rise to most of the future limb elements - bone, cartilage and tendon/ligament, has not been established. In this study, we developed the induction and expansion protocol of LBM cells from human iPSCs, and a high-throughput drug screening system using human LBM cells differentiated from iPSC which is derived from patients with skeletal dysplasia.

[Method] Paired-related homeobox 1 (PRRX1) serves as a marker for the LBM. To assess the induction efficiency of PRRX1 positive cells during each differentiation step, we established PRRX1-tdTomato reporter human iPSC line. Lateral plate mesoderm cells (LPM) derived from PRRX1-tdTomato reporter were used to establish the LBM-inducing method that recapitulates human developmental process. The chondrogenic capacity of expandable LBM (ExpLBM) cells was assessed using our two- or three- dimensional chondrogenic induction method (2DCI or 3DCI). In addition, ExpLBM cells differentiated from iPSCs derived from patients with type II collagenopathy (COL2pathy), one of the skeletal dysplasia arising from mutations in COL2A1, were used to develop high-throughput screening system.

[Results] By activating WNT signaling and inhibiting BMP/TGFb/headge hog signaling pathways, almost all LPM cells could be induced to PRRX1 positive LBM cells. Interestingly, we found the defined culture method that can not only stably expand LBM cells (ExpLBM) but also maintain their PRRX1 expression. ExpLBM formed Alcian Blue positive nodules under 2DCI condition and Safranin O positive cartilaginous particles under 3DCI condition. 2DCI-based high-throughput screening system found that several chemicals improved the chondrogenic capacity of ExpLBM derived from COL2pathy patient.

[Conclusion] ExpLBM cells will be a potential tool to study human bone development, cartilage regeneration and drug discovery using disease-specific human iPSCs.

We previously clarified that Extracellular signal-regulated kinase 5 (Erk5) directly phosphorylates Smad specific E3 ubiquitin protein ligase 2 (Smurf2) at Thr²⁴⁹ (Smurf2^Thr249), which leads to activating its ubiquitin ligase activity. Although we have demonstrated that Erk5 in embryonic mesenchymal stem cells (MSCs) is necessary for proper skeletogenesis, its role in adult bone marrow (BM)-MSCs on bone homeostasis remains unknown. It has been reported that Leptin receptor-positive (LepR⁺) BM-MSCs represent a major source of bone in adult bone marrow. Here, we unveiled the importance of Erk5 in BM-MSCs as a critical regulator of bone homeostasis in adulthood. Mice lacking Erk5 in LepR⁺ BM-MSCs showed osteosclerotic bone marrow with age in vivo. Erk5 deficiency increased differentiation of BM-MSCs toward osteoblasts along with a higher expression of transcription factors for osteogenic differentiation in vitro. Erk5 deficiency decreased Smurf2^Thr249 phosphorylation, and subsequently increased Smad1/5/8-dependent signaling in BM-MSCs. Genetic induction of the phosphomimetic mutant of Smurf2 rescued the osteosclerotic phenotype in Erk5-deficient mice. These findings suggest that the Erk5–Smurf2^Thr249 axis in BM-MSCs plays an essential role in maintaining proper bone homeostasis in adult bone marrow.

Achondroplasia (ACH) is a rare genetic disease where bone growth is abnormal and cartilage does not correctly form. The main characteristics of ACH are limb shortening and short stature. ACH is caused by single point mutations in the fibroblast growth factor receptor 3 (FGFR3), which leads to hyperactive receptor signaling. Cyclin-dependent kinase 8 (CDK8), which belongs to the transcription-related CDK family, has been reported to play an important role in regulating fundamental cellular processes including proliferation, survival, and differentiation. However, the functional role and underlying mechanisms of CDK8 on progression of ACH remain largely unknown. Here, we demonstrated that the pharmacological inhibition by CDK8 kinase inhibitor KY-065 significantly restores the impairment of chondrogenic differentiation and hypertrophy in mesenchymal cells derived from ACH model mice in vitro. Mechanistically, we found that KY-065 corrects the overactivation of STAT1 signaling via inhibition of CDK8 kinase activity, which in turn rescues the abnormalities of chondrogenesis in mesenchymal cells. Moreover, daily subcutaneous administration of KY-065 significantly increased bone growth of femur in ACH model mice. These findings indicate that CDK8 inhibition could be a potential therapeutic approach for ACH and other FGFR-related skeletal dysplasias.

Periodontal disease, which is majorly caused by Porphyromonas gulae, is extremely common in dogs, and is associated with serious systemic diseases such as kidney failure, heart disease and immune disorder. Ozon water is ozone-gas-dissolved water, which has antimicrobial activity by oxidizing molecules. However, both therapeutic and preventive effects of ozone water for periodontal disease in dogs are still unclear. We herein examined the antibacterial and anti-inflammatory effects of ozone water in vitro using P. gulae. Our results indicated that treatment of ozone water significantly inhibited　the growth of P. gulae until 24h post ozone water treatment as compared to purified water treatment. Ozone water also showed the anti-inflammatory effects in P. gulae-infected human gingival epithelial cells (Ca9-22). Secretions of IL-1β and TNFα were significantly suppressed by ozone water treatment compared to the treatment of purified water. As IL-1β and TNFα activate the osteoclast and finally promote bone resorption in the oral cavity, our findings imply the potential therapeutic and preventive effects of ozone water in canine periodontal disease. We are currently investigating the in vivo preventive and therapeutic effects of ozone water in dogs.

Intracellular Ca²⁺ ([Ca²⁺]_i) signaling has a critical role in osteoblast proliferation and differentiation. Membrane hyperpolarization increases the driving force for Ca²⁺ signaling and promotes osteogenic differentiation in human mesenchymal stem cells. Therefore, K⁺ channels are key regulators of osteoblast differentiation, however, the functional role of K⁺ channels in osteoblast Ca²⁺ signaling remains unknown. In the present study, the contribution of K⁺ channels to [Ca²⁺]_i mobility in the osteoblastic cell line MC3T3-E1, established from mouse calvaria was investigated. We found that the expression levels of inward rectifier K⁺ channel Kir2.1 transcripts and proteins were upregulated in the differentiated MC3T3-E1 cells. The application of ML133, a Kir2 inhibitor, significantly reduced the store-operated Ca²⁺ entry in differentiated MC3T3-E1 cells, and suppressed the expression of the differentiation markers in osteoblasts. The expression levels of Kir2.1 proteins were also upregulated in murine embryonic metatarsals, depending on the progression of the endochondral ossification. In addition, we showed that downregulation of miR-106b-5p augmented the expression levels of Kir2.1 in differentiated MC3T3-E1 cells. These results suggest that Kir2.1 channels play essential roles in maintaining the bone homeostasis via modulating osteoblast differentiation.

Tuberculosis (TB) patients must receive treatment for a long period, spanning at least 6 months. In some cases, this long period of treatment leads to inappropriate administration of anti-TB drugs and cessation of TB therapy, a hotbed of antimicrobial resistance. From this perspective, novel drugs that act synergistically or additively in combination with major anti-TB drugs are required to shorten the duration of TB therapy. Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is a powerful genetic tool which is expected to accelerate the development of TB drugs. In this study, we investigated whether CRISPRi could be used for predictive screening of the combined effect of anti-TB drugs. Knockdown of inhA, a target molecule of isoniazid (INH), increased susceptibility to rifampicin (RFP) and ethambutol, which act synergistically or additively with INH. This phenomenon was also true in the case of knockdown of rpoB, a target molecule of RFP. Moreover, CRISPRi could successfully predict the synergistic action of cyclomarin A with INH or RFP. These results demonstrate that CRISPRi is a helpful tool not only for exploring drug targets, but also for screening the combinatorial effects of known anti-TB drugs. This study provides evidence of CRISPRi platform-based anti-TB drug development.

Morphological study revealed that sympathetic neurons are closely associated with PDGFRα ⁺ cells in mouse colonic musculature. In addition, gene expression study showed that α1A adrenoceptors (α1A ARs) are expressed exclusively in PDGFRα ⁺ cells of mouse colon. Therefore, we investigated how sympathetic neurons regulate mouse colonic motility via α1A ARs in PDGFRα ⁺ cells. Noradrenaline (NAd), via α1A ARs, activated a small conductance Ca²⁺-activated K⁺ (SK) channels and hyperpolarized a single PDGFRα ⁺ cell (the α1A AR – SK channel signal pathway), resulting in hyperpolarization of neighboring smooth muscle cells (SMCs) connected by gap junctions, leading to inhibition of spontaneous contractions of colonic muscle. Sympathetic nerve stimulation (SNS) inhibited propulsive contractions represented by the colonic migrating motor complexes (CMMCs) via the α1A AR – SK channel signal pathway in wild type mouse, however CMMCs were not affected by SNS in the colon of Adra1a^−/− mouse. These results suggest that NAd released from sympathetic neurons inhibited mouse colonic motility via the α1A AR – SK channel signal pathway in PDGFRα ⁺ cells.

[Background and purpose]

Lactulose is a non-digestible galactose-fructose disaccharide, which is clinically used for the treatment of chronic constipation and hepatic encephalopathy. This study is aimed to clarify the therapeutic effect of lactulose on the intestinal flora and colitis associated tumor formation in a colitis-associated tumor mouse model.

[Method]

Model mice were created by intraperitoneal administration of 12 mg/kg BW azoxymethane (AOM) followed by 3 sets of sodium dextran sulfate (DSS) treatment by feeding 2% DSS-containing drinking water for 7 days. The lactulose treatment was started after DSS treatment, by feeding 2% lactulose-containing chow for 14 weeks. The metagenome analysis of feces, histopathological analysis and inflammation-related analysis were performed 24 weeks after AOM administration.

[Result]

In the AOM / DSS model, the greater degree of intestinal inflammation, fibrosis and colorectal tumorigenesis as well as alteration of the structure of intestinal flora were observed compared to the normal group. The mRNA expression of TNF-α, IL-6, and TGF-β1 at non-tumor areas was upregulated. Lactulose treatment significantly improved the inflammation and tumorigenesis. Lactulose treatment restored the structure of intestinal flora, especially with respect to Bacteroides caecimuris, Lactobacillus intestinalis, and Lactobacillus murinus. 

[Discussion]

The suppression of inflammatory bowel tumor formation by lactulose correlates with improvement of the intestinal flora structure.

Aim: We focused on eosinophilic esophagitis (EoE), a refractory rare disease, and tried to find the feature of oral microbiome in EoE patients and relationship between infection of pathogenic oral bacteria and EoE.

Method: Fifty healthy control volunteers, 52 EoE subjects were recruited. Bacterial DNA was extracted from the mouse-wash of subjects. Then, four popular periotontal pathogens (P.g, Prevotella intermedia (P.i), Tannerella forsythia (T.f), Treponema denticola (T.d)) and one caries-causing bacteria (S.m) were detected by PCR with specific paired-primer for each bacterium. Oral microbiome was analyzed by new generation sequencing. Furthermore, we examined correlationships between population or infection of bacteria and EoE-related parameters including number of eosinophils in esophagus, percentage of eosinophils in peripheral blood and serum IgE value.

Resutls: Infection of T.f and S.m were strongly correlated with EoE. Diversity of oral bacteria was smaller in EoE compared to that in healthy control. Only population of Neiserria mucosa was negatively correlated with number of eosinophils in esophagus although we examined all combination.

Conclusion: EoE subjects seems to have less abundant microbiome and higher infection rate of T.f and S.m. These changes possibly affect disease state of EoE.

Cells are often exposed to hypoxic condition in microenvironment of our body. Hypoxic condition induces various adaptive responses including alteration of metabolism, respiration, and cell growth. Furthermore, hypoxia induces malignant transformation of cancer cells, and promotes invasion and metastasis. Hypoxia-Inducible Factor (HIF) is a transcription factor which plays a key role in hypoxic response by inducing expression of multiple genes. Previously, we have demonstrated that HIF is activated upon early phase of hypoxic condition, whereas CREB becomes activated during late phase of hypoxia when HIF is downregulated.

In addition to transcription factors, chromosome conformation is a critical factor to determine the gene expression status. To understand the significance of higher order genome organization in the regulation of hypoxic gene expression, we have performed a large scale nuclear gene position analysis of hypoxia-responsive genes. Using high-throughput imaging, radial and relative nuclear positions of more than 100 HIF- and CREB-target genes were mapped in 3D space under hypoxic condition. We find that that some genes changed their nuclear positions and their relative positions to each other in response to hypoxic treatment for 48 h whereas others did not. However, no pervasive correlation between the distance and gene expression was observed. Possible roles of gene repositioning under hypoxic condition will be discussed.

Plasma irradiation not only produces a variety of reactive species, but can also modify the molecular structure of materials at the atomic level. In recent years, it has been focused on the effects of specific chemical reactions induced by low-temperature plasma irradiation on biological activities, such as sterilization, wound healing, and anti-tumor effects. However, the molecular mechanisms of these effects have remained unclear. We found that irradiation of non-equilibrium atmospheric pressure plasma with humidified helium gas into cysteine (Cys-SH) solution produces sulfur metabolic intermediates such as cysteine persulfide (Cys-SSH) and thiosulfate ion. We previously showed that reactive sulfur species (RSS) such as Cys-SSH play an important role in mitochondrial energy metabolism and that RSS metabolism contributes to ischemic tolerance. Plasma-irradiated cysteine solution (Cys^*) significantly suppressed mitochondrial dysfunction in hypoxic cardiomyocytes and myocardial cell death induced by reoxygenation. The accumulation of sulfide and intracellular RSS catabolism under hypoxia was significantly inhibited by Cys^*. Furthermore, injection of Cys^* into mice improved cardiac function after ischemia/reperfusion injury. These results suggest that Cys^* containing various sulfur intermediates exerts a cardioprotective effect by negatively regulating intracellular RSS catabolism in the absence of O₂ and by maintaining energy metabolism in mitochondria.

Glycolysis plays an important role in cell homeostasis and growth through not only producing ATP but also supplying glycolytic intermediates to biosynthetic pathways. However, how glycolysis is regulated during cardiac hypertrophy is unknown. Yes-associated protein 1 (YAP), a major transcriptional cofactor in the Hippo signaling pathway, is known to regulate cell growth and energy metabolism. Here we examined whether YAP regulates glycolysis in cardiomyocytes. Glucose stress test was performed in neonatal rat ventricular myocytes (NRVMs) transduced with adenoviruses harboring LacZ or YAP. Overexpressed YAP significantly increased glycolytic function and metabolic intermediates of the glycolytic pathway and upregulated both mRNA and protein levels of glucose transporter 1 (Glut1), thereby stimulating glycolysis. Knockdown of Tead1 or Hif-1α, but not c-Myc, inhibited the YAP-induced upregulation of Glut1 protein in NRVMs. YAP-S94A mutant, which is unable to interact with TEAD1, failed to upregulate Glut1 and increase glycolysis. ChIP assays revealed that YAP, Tead1, and Hif-1α bind to the Glut1 promotor in cardiomyocytes. These results suggest that YAP promotes cardiomyocyte glycolysis through a YAP-TEAD1-HIF-1α-dependent transcriptional upregulation of the Glut1 promoter activity.

【Background and Objective】

Since mammalian cardiomyocytes exit from cell cycle immediately after birth, the regenerative activity is limited in adult mammalian hearts. The aim of this study is to conduct a comprehensive search for pro-proliferative factors in mammalian cardiomyocytes, using a Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) that labels S/G2/M phases nuclei in green.

【Methods and Results】

Neonatal rat heart-derived cells were infected with the adenovirus vector expressing FUCCI or GFP. Immunofluorescence microscopic analysis showed that more than 95% of FUCCI or GFP positive cells represented cardiomyocytes. Next, FUCCI or GFP positive cells were obtained using FACS and analyzed by RNA-seq. One hundred and eight genes, including pro-proliferative factors such as various cyclins, were increased in FUCCI positive cells compared with GFP positive cells. Interestingly, we found that the expression of several genes related with cancer cell proliferation was also upregulated in FUCCI positive cells.

【Conclusion】

A system to comprehensively search for cell proliferation-related factors in cardiomyocytes was constructed using FUCCI expressing adenovirus and FACS.

Microsomal prostaglandin E synthase-1 (mPGES-1)/prostaglandin E₂ (PGE₂) induces angiogenesis through the prostaglandin E₂ receptor (EP1­–4). PGE₂ is not only known to induce angiogenesis but also known to modulate function and differentiation of regulatory T cells (Tregs)_. We evaluated effect of Tregs on angiogenesis by using acute hind limb ischemia model.

Male 6-8 week-old wild-type mice (WT), mPGES-1-deficient mice (mPges-1^-/-) and EP4 recepor deficient mice (EP4^-/-) were used. Recovery from ischemia was estimated by laser Doppler imaging. Angiogenesis was estimated by CD31 and TGF-beta and by using immunohistochemical analysis and real time PCR. Contribution of Tregs was estimated by immunohistichemical study and real time PCR against FoxP3.

Compared to WT, blood flow recovery was significantly suppressed in mPGES-1^-/-. The blood flow recovery was significantly enhanced by EP4 agonist but suppressed by EP4 antagonist. EP4^-/- significantly suppressed blood flow recovery compared to EP4. CD31⁺ cells and TGF-beta mRNA expression in ischemic muscle were significantly decreased in EP4^-/- mice compared to WT. The number of accumulated FoxP3+ cells and Foxp3 mRNA expression were significantly decreased in EP4^-/-. Moreover, blood flow recovery of Ep4^-/- was significantly improved by transplantation of CD4⁺ cells, containing Tregs, from WT, whereas infusion of Tregs-depleted CD4⁺ cells did not restore blood flow in Ep4^-/-.

These findings suggested that mPGES-1/PGE₂ induced blood flow recovery from ischemia via EP4 by promoting accumulation of Tregs.

Endothelial cells sense extracellular substrate stiffness and adapt their function. Vascular or arterial stiffening observed during hypertension, arteriosclerosis, and acute inflammation may be involved in endothelial cell dysfunction; however, the impact of substrate stiffness on endothelial cell functions remains elusive. First, we confirmed that endothelial cells cultured on softer substrates not only elongate cellular aspects but also attenuate yes-associated protein 1 (YAP) activation compared to cells on stiffer substrates. Endothelial cells on softer substrates upregulated the vascular endothelial growth factor receptors mRNA expression. Next, we found that endothelial cells on softer substrates induced delta-like ligand 4 (Dll4) expression via YAP1 deactivation and Notch1 activation. Moreover, endothelial cells on soft substrates induced suppression of pro-inflammatory interleukin-6 and plasminogen activator inhibitor-1 mRNA expression and the facilitation of anti-coagulant thrombomodulin and pro-coagulant tissue factor mRNA expression, all of that partially regulated by the YAP-Dll4-Notch pathway. Our results indicated that endothelial cells modulate cell functions in response to substrate stiffness through activating the YAP-Dll4-Notch pathway. These suggested that vascular or arterial stiffening promotes endothelial cell dysfunction leading to be the onset of vascular inflammatory diseases.

Adolescent binge drinking represents a major public health challenge and lead to persistent psychiatric disorders. However, the underlying pathogenic mechanisms remain poorly understood. Myelin abnormalities were observed in human subjects with alcohol abuse. Gray matter myelination in the prefrontal cortex and hippocampus during adolescence are vulnerable to alcohol.In the present study, we investigated whether myelin abnormalities in the gray matter are involved in behavioral abnormalities induced by adolescent binge ethanol treatment (ABET). To produce ABET, C57BL/6J mouse was given EtOH (3.0g/kg, i.e. 25% ethanol w/v) once a day during adolescence (P28-46) in an intermittent fashion. ABET persistently developed behavioral abnormalities such as anxiety-like behaviors in the marble burying test and the novelty suppressed feeding test, impaired memory function in the novel object recognition test, and impairments of social behavior in social interaction test. ABET decreased myelin-related protein and oligodendrocyte lineage cells in the prefrontal cortex and hippocampus. Clemastine, which promotes oligodendroglial differentiation and myelination, rescues the behavioral abnormalities. These findings suggest that myelin abnormalities in the gray matter may be involved in the behavioral abnormalities caused by ABET in adulthood.

Orofacial neuropathic pain dampens QOL by disturbing daily activates including chewing and talking. Accumulating evidence indicates the importance of cytokines, such as interleukin (IL)-1 beta and IL-18, in the development of neuropathic pain; however, the role of IL-33, a member of IL-1 family, in neuropathic pain has not been fully understood. Here, we investigate the role of IL-33 in orofacial neuropathic pain using infraorbital nerve injury (IONI) mice. Mechanical allodynia in the whisker pad skin and an increase in the amount of IL-33 in the Vc were observed following IONI. IL-33 and its receptor were exclusively expressed in oligodendrocytes and neurons, respectively. IONI-induced mechanical allodynia was ameliorated by neutralizing IL-33 in the Vc. Conversely, intracisternal administration of IL-33 triggered mechanical allodynia in naïve mice without affecting glial activation. Intracisternal administration of IL-33 facilitated phosphorylation of GluN2B, a subunit of NMDA receptor, in the synaptosomal fraction from the Vc and potentiated GluN2B-mediated miniature excitatory postsynaptic currents in the Vc. Furthermore, a specific GluN2B antagonist reversed IL-33-induced mechanical allodynia. Intracisternal administration of IL-33 caused phosphorylation of Fyn kinase but not Src kinase. Following IL-33 administration, phosphorylated Fyn kinase was located in the dendritic spine of the Vc. Inhibition of Fyn phosphorylation decreased IL-33-induced phosphorylated GluN2B in the Vc and prevented IL-33-induced mechanical allodynia. These results suggest that oligodendrocytes-derived IL-33 activates Vc neurons by phosphorylation of Fyn kinase and subsequent phosphorylation of GluN2B, culminating in the orofacial neuropathic pain. Our results lead to the development of orofacial neuropathic pain targeting the IL-33 signaling.

Despite several lines of evidence indicate that spinal microglia participate in pain processing, it is pivotal to know whether there are differential roles of microglia in pain hypersensitivity between male and female mice. Induction of Gq-DREADD in spinal microglia elicited mechanical allodynia in male mice, but not in female mice, through the upregulation of inflammatory molecules. Peripheral nerve injury-induced mechanical allodynia was suppressed by the treatment of PLX3397, a microglial inhibitor, in male but not in female mice. Importantly, the effects of microglial activators and inhibitors (i.e., Gq-DREADD and PLX3397) in the spinal dorsal horn of male mice was significantly greater than that of female mice. Moreover, nerve injury-induced mechanical allodynia in female mice was prevented by administration of testosterone propionate, suggesting that androgens might determines sex differences of microglia. Collectively, male microglia have a potential to exacerbate pain sensitivity in mice, and further study on androgen actions may provide the key mechanisms underlying sex differences in spinal microglia.

Central post-stroke pain (CPSP) is a type of neuropathic pain for which the mechanisms and relevant drug pathways remain unknown. Previously, we have demonstrated that bilateral carotid artery occlusion (BCAO) induced CPSP model mice showed mechanical hypersensitivity, and the intracerebral (i.c.v.) injection of nicotine suppress this phenomenon. However, the detailed mechanisms of it are not fully understood. Recently, nicotine regulates the neuronal activity of orexin in the lateral hypothalamus (LH). In this study, we evaluated the mechanism underlying the antinociceptive effect of nicotine. Male ddY mice were subjected to 30 min of BCAO. Mechanical hypersensitivity was assessed by von Frey test. BCAO mice showed hypersensitivity against mechanical stimuli at 3 days after BCAO operation. The i.c.v. injection of nicotine (20 nmol) suppressed BCAO-induced mechanical hypersensitivity. After nicotine injection, the number of c-fos, a neuronal activity marker, increased in the LH and the locus coeruleus (LC) in Sham and BCAO mice. These increments were colocalized in orexin A positive cells in the LH or tyrosine hydroxylase positive cells in the LC. Nicotine-induced antinociception was inhibited by the intrathecal pretreatment of yohimbine, a a2 adrenergic receptor antagonist. These results indicate that nicotine may suppress BCAO-induced mechanical hypersensitivity through activation of the descending pain control system via facilitating orexin-A/orexin receptor 1 receptor signaling.

Pain transmission in the spinal dorsal horn (SDH) is powerfully controlled by descending neurons from the brain. One major neurotransmitter of the descending pathways is noradrenaline (NA). Traditionally, NA is known to produce an analgesic effect, but we recently demonstrated that NA also induces a pronociceptive effect via α_1A-adrenoceptors (α_1A-ARs) in SDH astrocytes expressing Hes5. Therefore, the mechanism for bidirectional modulation by NA is entirely unknown. In this study, we found that intrathecal injection of NA at a low dose induced mechanical hypersensitivity, which required α_1A-ARs in Hes5⁺ astrocytes. On the other hand, at a high dose of intrathecal NA, the hypersensitivity disappeared. Interestingly, this effect of the high dose of NA was not affected by lacking α_1A-ARs in Hes5⁺ astrocytes, but was prevented by pharmacological inhibition of spinal β₁-ARs or genetic knockout of astrocytic β₁-ARs. Furthermore, mechanical hypersensitivity by chronic stress was suppressed and exacerbated by astrocytic α_1A- and β₁-AR-knockout, respectively. Our findings suggest that the bidirectional pain control by spinal NA is dependent on the levels of NA and the different types of astrocytic adrenoceptors.

Ryanodine receptor 2 (RyR2) is a Ca²⁺-release channel on the sarcoplasmic reticulum of cardiomyocyte that plays a central role in cardiac excitation-contraction coupling. Spontaneous Ca²⁺ release events caused by abnormal hyperactive RyR2 are linked to ventricular arrhythmias in patients with heart failure and catecholaminergic polymorphic ventricular tachycardia (CPVT). To search for novel RyR2 inhibitors from well-characterized drug library, we performed a high-throughput screening using HEK293 cells that stably express WT RyR2 and R-CEPIA1er, a genetically encoded endoplasmic reticulum (ER) Ca²⁺ indicator. By screening 1,535 compounds, we identified three ones that increased the ER Ca²⁺ signal greater than 4SD above the mean: chloroxylenol (Clxy), orserinate β-methyl (OBM) and riluzole (Ril). All three compounds increased ER Ca²⁺ signal and decreased the frequency of spontaneous Ca²⁺ oscillations dose-dependently in HEK293 cells expressing CPVT-linked mutant RyR2s as well as WT RyR2, which corresponded reduction of the Ca²⁺-dependent [³H]ryanodine binding. To investigate the drug effects on arrhythmogenic Ca²⁺ release, we observed Ca²⁺ signals in cardiomyocytes obtained from Tnnt2 ΔK210 and RYR2 I4093V mice that show Ca²⁺ waves, sparks and triggered activity frequently. Ril and Clxy suppressed Ca²⁺ waves without affecting action potential induced Ca²⁺ transients. These results suggest that the RyR2 inhibitors are promising candidates for novel anti-arrhythmic drugs.

Loss-of-function (LOF) mutations in type 2 ryanodine receptor (RyR2) have been linked to various types of cardiac arrhythmias including idiopathic ventricular fibrillation (IVF), short-coupled variant of Torsades de Pointes (scTdP) and long QT syndrome (LQTS). However, cellular mechanisms of divergent phenotypes remain unclear. We investigated Ca²⁺ signaling by four LOF RyR2 mutants, E4146D (ED), S4168P (SP), K4594Q (KQ) and S4938F (SF), in HEK293 cells and HL-1 cardiomyocytes. Patients with ED and SF showed VF and scTdP, whereas those with SP and KQ showed LQTS without life-threatening events. ED and SF moderately suppressed [³H]ryanodine binding with reduced Ca²⁺ sensitivity, and SP and KQ almost completely suppressed the binding. In HEK293 cells which express homotetrameric RyR2s, WT cells showed cytosolic ([Ca²⁺]_cyt) oscillations with concomitant decrease in ER Ca²⁺ ([Ca²⁺]_ER), but all the LOF mutant cells showed elevated [Ca²⁺]_ER with no [Ca²⁺]_cyt oscillations. In HL-1 cells, which have endogenous WT RyR2s, expression of SP or KQ exerted no Ca²⁺ waves but only reduced Ca²⁺ transients. In contrast, ED and SF exerted frequent localized Ca²⁺ waves. These results suggest that moderate suppression of RyR2 may cause abnormal Ca²⁺ signaling leading to scTdP and VF, whereas severe suppression results in less arrhythmic events. Ca²⁺ signaling by heterotetrameric channels composed of WT and mutant RyR2s need to be further clarified.

We have shown that the aortic elasticity was increased by hypertensive doses of angiotensin II (ATII) in contrast to the decrement of femoral arterial elasticity. To clarify the regulatory mechanisms of arterial elasticity, we analyzed effects of various types of cardiovascular drugs on arterial elasticity in rabbits using the stiffness parameter β of the aortic (aortic β) and femoral arterial (femoral β) regions by measuring blood pressures at the right brachial artery, aortic bifurcation and tibial artery, electrocardiogram and phonocardiogram. Vasoconstrictor drugs ATII (300 ng/kg) and phenylephrine (10 µg/kg) as well as a cardiotonic drug dobutamine (30 µg/kg), each of which increased blood pressure by about 20 mmHg, decreased the aortic β and increased the femoral β. Vasodilator drugs nifedipine (300 µg/kg) and cilnidipine (30 µg/kg) as well as a cardiodepressor drug metoprolol (10 µg/kg), each of which decreased blood pressure by 10-15 mmHg, increased the aortic β and tended to decrease the femoral β. These results suggest that femoral arterial elasticity may be regulated by the pharmacological action of each drug. On the other hand, aortic elasticity may be regulated by pressure-sensitive intrinsic mechanism of aortic wall to optimize aortic elasticity, independently of pharmacological mechanisms of action of each drug.

Nardilysin (NRDC; N-arginine dibasic convertase) is a metalloprotease of the M16 family. We have shown that NRDC has multiple functions such as an enhancer of ectodomain shedding of membrane-anchored protein and transcriptional coregulator. NRDC-deficient mice (Nrdc^-/-) show wide range of phenotypes such as hypomyelination, hypothermia, abnormal sympathetic innervation to hearts, hypotension, and bradycardia. The purpose of this study is to explore a role of NRDC in the regulation of heart rate. To this end, we have revealed the following points: (1) Intrinsic heart rate, obtained by pharmacological blocking of autonomic nervous system, was significantly reduced in Nrdc^-/-　compared with that of wild-type mice; (2) Messenger RNA levels of Cav3.1 and HCN1/4, ion channels responsible for sinus automaticity, were significantly reduced In Nrdc^-/- hearts; (3) Funny (If) current and T-type Ca current were markedly reduced in Nrdc^-/- sinus node cells; (4) Gene knockdown of NRDC in primary rat cardiomyocyte reduced mRNA levels of HCN1/4; (5) NRDC binding to the promoter region of Cav3.1 and HCN1/4 was revealed by chromatin immunoprecipitation-PCR analysis, suggesting the direct involvement of NRDC in transcriptional regulation of these ion channels; (6) Reintroduction of wild-type NRDC, but not the enzymatic inactive mutant of NRDC (E>A mutant) into NRDC-deficient cells restored the HCN1 mRNA expression, suggesting the important role of NRDC enzyme activity in the transcriptional regulation; (7) NRDC-E>A mutant knock in mice showed bradycardia and significantly reduced intrinsic heart rate. Together, our results indicate that NRDC in cardiomyocyte controls heart rate through the transcriptional regulation of ion channels critical for sinus automaticity.

Introduction: TASK-1 channel, one of the two-pore domain potassium channels, is expressed mainly in the atria, inhibition of which may prolong the action potential duration of atrial myocyte. To investigate whether TASK-1 inhibition suppresses atrial fibrillation (AF), we studied the electropharmacological and anti-AF effects of a selective TASK-1/3 inhibitor doxapram in vivo.

Methods: Exp.1) Doxapram hydrochloride hydrate in doses of 0.3 and 3 mg/kg was intravenously administered over 10 min to the isoflurane-anesthetized intact dogs (n=6) to assess the electropharmacological effects. Exp.2) The same doses were infused to the isoflurane-anesthetized, chronic atrioventricular block dogs (n=5) to assess the antiarrhythmic effect against paroxysmal AF induced by 10 s of atrial burst pacing.

Results: Exp.1) Doxapram showed positive chronotropic, inotropic and dromotropic effects and delayed the ventricular repolarization, whereas it hardly altered the atrial or ventricular conduction velocity. Doxapram prolonged the atrial effective refractory period (AERP), which did not exhibit frequency dependency. Exp.2) The low and high doses of doxapram tended to rather prolong the AF duration by +22 and +26% from the pre-drug control, respectively.

Conclusion: Doxapram did not suppress the paroxysmal AF in vivo, which may be associated with the lack of atrial conduction delay and the less great prolongation of AERP at faster atrial pacing rate. These results suggest that TASK-1 inhibition might have limited efficacy on AF prevention and termination.

Amyloid-β (Aβ) peptides are produced from the sequential cleavage of the β-amyloid precursor protein (APP) by the β-site APP cleaving enzyme 1 (BACE1) and γ-secretase. Since Aβ accumulates in the brains of patients with Alzheimer's disease (AD), attempts to develop therapies based on the "amyloid hypothesis" have been made. However, many of them have now failed. Therefore, there is a need to develop a new hypothesis that complements the amyloid hypothesis. Interestingly, dysfunction of endosomes, which mediate the vesicular trafficking, has been shown to be one of the earliest pathophysiologies in AD before Aβ accumulation. So the "traffic jam hypothesis," in which vesicular traffic impairment is the trigger of AD pathogenesis, is attracting attention. However, the mechanistic details remain unclear.

We have previously shown that β-carboxyl-terminal fragment of APP (βCTF), a direct precursor of Aβ, binds to TMEM30A, a component of lipid flippases. These enzymes translocate phospholipids from the exoplasmic/luminal side to the cytoplasmic leaflet of the lipid bilayer to regulate phospholipid asymmetry, and important for regulating vesicular trafficking. We have found that binding of βCTF reduces lipid flippase activity and induces vesicular traffic impairment. Furthermore, we identified these disorders are ameliorated by the βCTF-binding peptide "T-RAP."

In this session, we will discuss the development of a method to screen therapeutic drug candidates that bind to βCTF by applying T-RAP.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder marked by the accumulation of amyloid-β (Aβ) plaques and hyperphosphorylated tau tangles. Thus, noninvasive detection of accumulated Aβ and tau in the brain would be beneficial for an early diagnosis of AD. We developed a novel near-infrared fluorescence (NIRF) probe named THK565 for in vivo imaging of Aβ and tau in the brain. In the present study, we evaluated whether THK565 can noninvasively detect Aβ and tau deposits using 2 lines of AD-model mice. We used APP-KI and rTg4510 mice which show progressive accumulation of Aβ and tau in the brain, respectively. Mice were intravenously injected with 0.3 mg/kg of THK565, and then in vivo fluorescence signals were measured using IVIS imaging system at different time point. After in vivo imaging, we harvested brain and confirmed the existence of Aβ and tau deposits by immunohistochemistry.

In vivo NIRF imaging demonstrated higher fluorescent intensity in the brains of APP-KI and rTg4510 than in those of age-matched control mice. The histological analysis confirmed that THK565 clearly labeled Aβ and tau plaques in the brain section. These findings imply that THK565 is useful for noninvasive assessment of Aβ and tau pathology in the brain.

Parkinson's disease is a neurodegenerative disease characterized by the degeneration of dopamine neurons in the substantia nigra. ATP13A2 has been reported as a causative gene of PARK9. ATP13A2 is a lysosomal localized ATPase and involved in the transport of metal ions such as divalent iron ions (Fe²⁺). In addition, iron accumulation is observed in the brains of PARK9 patients. Therefore, it is considered that ATP13A2 contributes to the maintenance of intracellular iron homeostasis. However, there are still unclear points about the effect of ATP13A2 dysfunction on intracellular iron homeostasis and the mechanism of neurodegeneration. To reveal these issues, a PARK9 model cell was created by ATP13A2 knockdown in SH-SY5Y cells. In addition, their effects on lysosomal function and intracellular iron homeostasis were analyzed. As a result, an increase in oxidative stress and a decrease in cell viability were observed in PARK9 model cell. In addition, autophagy markers and α-synuclein were increased in PARK9 model cell, suggested that ATP13A2 knockdown induced autophagy dysfunction. Moreover, accumulation of Fe²⁺ in lysosomes and mitochondria was confirmed by fluorescent probes in PARK9 model cell, indicating that ATP13A2 knockdown caused the disruption of intracellular iron homeostasis. These results contribute to reveal the pathophysiology of Parkinson's disease and diseases related to iron deposition.