Endothelial dysfunction is a key factor in development of diabetic vascular complications. We and others reported that vegetable polyphenol morin (MO) and quercetin (QC) induced vascular endothelial relaxations through the Akt/eNOS signaling pathway. However, the relaxation pathway was fully unknown. In this study, we examined about the upstream of the Akt/eNOS signaling in diabetes. In aortas isolated from diabetic (DM) or control mice, MO and QC induced dose-dependent relaxation responses. Especially, DM aortas showed enhanced MO-induced relaxation responses relative to controls. The relaxation responses with MO were not significant in the presence of each PI3K inhibitor and AMPK inhibitor. Meanwhile, the relaxation responses with QC were attenuated in the presence of PI3K inhibitor. In the presence of AMPK inhibitor, the relaxation responses with QC were attenuated in DM but there were not significant in control. In this study, we showed that QC induced NO-dependent relaxation responses via PI3K/Akt/eNOS signaling pathway in control, notably AMPK/Akt/eNOS signaling in DM. Thus, it was suggested the possibility that MO and QC have different mechanism in relaxation, and that MO had the beneficial effect on the diabetes induced endothelial dysfunction.

Abnormal G-protein-coupled receptor kinase 2 (GRK2) accumulation has a crucial role in the development of insulin resistance and diabetes. Previous report showed that impaired insulin-induced relaxation in diabetes is improved by suppressing the GRK2 levels. Glucagon-like peptide-1 (GLP-1) is a gut hormone that promote insulin secretion. However, it is unknown whether GLP-1 directly affects diabetic endothelial dysfunction, especially GRK2 signaling. In this study, we investigated the relationship between GLP-1 and GRK2 under insulin stimulation for endothelial dysfunction. GLP-1 increased the impaired insulin-induced NO-dependent relaxation responses in diabetes. However, these responses are disappeared by treatment of Exendin-9, a GLP-1 receptor antagonist. Furthermore, phosphorylation levels of Akt and eNOS were higher in diabetes under GLP-1/insulin stimulation than non-stimulation. Additionally, in diabetes GRK2 activity was inhibited under GLP-1/insulin stimulation compared with non-stimulation, although GRK2 expression was not altered between the two groups. Those results suggest that in diabetes, GLP-1 improves the endothelial dysfunction by suppressing of GRK2 activity, which might provide a therapeutic target for diabetic vascular complications.

Protein kinase C (PKC) family plays crucial roles in a wide variety of cellular functions and dysregulation of PKC signaling leads to pathophysiological states including cancer and neurodegenerative disease. PKC activity is rigorously fine-tuned by multiple mechanisms, but the spatial regulatory mechanism of PKC signaling remains fully understood. Here, we have shown that RNA granules, widely conserved non-membranous cytoplasmic structures composed of RNA-protein complexes, play a key role in spatially regulating PKC and its downstream MAPK signaling activation. Upon heat stress, PKC and downstream MAPK signaling activation was induced, which stimulates recruitment of the PKC homologue in fission yeast, from the plasma membrane into RNA granules. Intriguingly, Inhibition of the downstream MAPK signaling impaired PKC translocation to the RNA granules, whereas the constitutively active MAPKK stimulated PKC translocation to the RNA granules. We also demonstrated that the kinase activity of PKC influenced its intracellular distribution from the cell membranes to the RNA granules.  Our data is the first demonstration that PKC translocation into RNA granules is a novel feedback mechanism mediated by MAPK signaling.

It is generally accepted that Propionibacterium acnes (P. Acne) is involved in the development of acne, while the mechanisms of sebaceous lipogenesis and its control is unclear. Enterococcus faecalis FK-23 had shown to promote an anti-inflammatory action in several animal models. In the current study, we examined whether FK-23 modulate lipogenesis in sebocytes. FK-23 stimulated lipogenesis, while inhibited them in the presence of P. Acne. FK-23 acutely inhibited acetyl-CoA carboxylase (ACC) phosphorylation levels, while stimulated them with P. Acne. FK-23 stimulated PPARγ expression and activity, while inhibited them with P. Acne as pioglitazone did. These combined evidences demonstrated that the dual action by FK-23 on lipogenesis should reflect differentiation machinery prior to PPARγ, leading to producing adequate levels of sebum.

Nε-(carboxymethyl) lysine (CML), an advanced glycation end product (AGE), is an aging factor produced by glycation of protein. Higher levels of AGE in skin tissue are related to skin elasticity, but how CML that has accumulated in the skin affects hair follicle formation is unclear. This study constructed a simple model that mimics accumulated glycation from feeding by intradermally injecting Nε-(carboxymethyl) lysine (CML), and examined the effects on the morphogenesis of hair follicles (HF). The results showed weakening of the hair shaft and HF formation by CML. The in vitro inhibitory effect of CML on wound healing of dermal papilla cells (DPC) suggested that the mechanism influences the proliferation and migration of DPC, which are essential for HF morphogenesis. In addition, CML in DPC inhibited the expression of sonic hedgehog (Shh), a factor of tissue morphogenesis, in a NF-kB-independent manner. The findings suggest that the delay in HF formation was due to CML inhibiting proliferation and migration in DPC by inhibiting Shh expression.

Amino acids in cells are used not only as building blocks for protein synthesis but also for other purposes such as signal transduction and ATP synthesis. For example, amino acids are essential signaling molecules to activate mTORC1 (mechanistic target of rapamycin complex 1), a serine/threonine kinase complex, which plays a key role in the regulation various biological processes including lipid synthesis, translation, autophagy and so on. Also, dysregulation of amino acid homeostasis is often implicated in the pathogenesis of cancer, diabetes and neurodegenerative disorder. Amino acids in cell culture medium are supposed to significantly affect the intracellular amino acid contents, and subsequently, amino acid-related cellular functions. However, the relationship between amino acid availability and intracellular amino acid concentration is still poorly understood. In this study, we established a reversed-phase HPLC method to quantitatively analyze the amino acids in cell lysates. Among 20 proteinogenic amino acids, 19 amino acids except tryptophan was successfully detected. By manipulating the amino acid availability in mammalian cell culture, the effects on the intracellular free amino acid concentration were further studied.

Skeletal muscle atrophy impairs quality of life. However, there is no effective medications. To maintain muscle mass, it is crucial to enhance differentiation of skeletal muscle stem cells and protein synthesis in myofibers. Our search using C2C12 cells determined that DCEBIO, a small/intermediate conductance Ca²⁺ activated K⁺ (SK/IK) channel opener promotes myogenic differentiation and muscle hypertrophy. DCEBIO hyperpolarized myoblast membrane potential and increased intracellular Ca²⁺ concentration. The expression of myogenin was elevated. DCEBIO treatment increased phosphorylation level of S6K, but not that of Akt. DCEBIO did not alter the expression of atrophy and hypertrophy related genes. SK/IK channel blocker, apamin (100 nM) and TRAM-34 (1 μM) inhibited myogenic differentiation but did not attenuate hypertrophic effect of DCEBIO in C2C12 cells. These results suggest that DCEBIO enhances myogenic differentiation by opening of SK/IK channel and activating myogenic regulatory factor. In contrast, DCEBIO increases muscle mass by activating S6K independent of SK/IK channel activation. This is a significant study for future drug development for muscle atrophy.

Social defeat stress develops depressive-like behaviors in mice by inducing inflammation both in the central nervous system and in the systemic circulation. Recent evidences have shown that the disruption of blood brain barrier leads to stress-induced behavioral alterations, suggesting that the integrity and deterioration of barriers surrounding the brain parenchyma is critical in stress-related pathophysiology. Although there has been emerging evidence showing the biochemical difference between the CSF of patients of depression and that of healthy controls, the effects of stress on the CSF system have rarely been studied. In the present study, we examined effects of social defeat stress on leptomeninges, which serve as barriers separating the brain parenchyma from the CSF and as regulators of immune reactions. We found an evidence of immediate phosphorylation of extracellular signal-regulated kinase (ERK) in the leptomeninges following a single episode of social defeat stress. This activation of ERK was diminished after repeated exposure to daily stress for 10 days. These evidences suggest that the leptomeninges are activated following an acute stress and diminish their responsiveness during the course of chronic stress.

[Purpose] We investigated the effect of (-)-nicotine (NIC) on social behaviors in the stressed model mice, and the expression of nicotinic acetylcholine receptor (nAChR) subunits or their intracellular signal-related molecules.

[Methods] The adult male C57BL/6J mice were exposed to forced swimming stress for 15 min.  They were treated with NIC (0.3 mg/kg s.c.) for 7 days from the next day of exposure to stress.

[Results] The stressed mice showed the impairment of social behaviors in the social interaction test and the decreased expression of phosphorylated Akt protein in the hippocampus (HIP).  These abnormalities were attenuated by repeated treatment with NIC. The expression of α7 and β2 nAChR subunit proteins was decreased in the HIP of the stressed mice.  α7, but not α4β2 nAChR antagonist blocked the ameliorating effect of NIC on behavioral impairment in the stressed mice.

[Conclusions] These results suggest that repeated treatment with NIC is useful and effectively for stress-induced depressive-like behaviors. The remission of social behavior impairment by NIC may be mediated via regulating nAChR/Akt signaling in stressed mice.

In order to address the mechanisms of stress response of the brain, it is important to characterize each stress responsive neuron. Recently, we performed whole brain imaging at single cell resolution in Arc-dVenus reporter mice, which express destabilized Venus in activated neurons. In these mice exposed to a stressor, dVenus signals were observed in a small brain region that strongly contributes to classifying stress and control brains. Here, we examined gene expression profiles of excitatory neurons in this region and compared stress responsive neurons with their neighboring non-responsive neurons using single-cell RNA sequencing. In Arc-dVenus mice, excitatory neurons were labeled by red fluorescent protein tdTomato using the AAV-PHP.eB vector. After the mice were subjected to a single social defeat stress, tdTomato-positive and dVenus-positive neurons were individually picked up and their gene expression profiles were compared. We identified the genes of which expression are induced by the stress and those that potentially classify the stress-responsive neurons from the non-responsive neurons. This study contributes to the understanding of the molecular basis of stress response and may open the door for specific analysis of stress responsive neurons.

We developed the fibromyalgia-like chronic pain model in mice by using intermittent cold stress. This model mouse was found to have similarity to fibromyalgia patients in terms of pathophysiology and pharmacotherapy, which includes the loss of sensitivity to morphine. Based on the speculation that excess amounts of endogenous opioids induced by repeated stress may cause a type of opioid analgesic tolerance, we attempted to use anti-opioid NMDA receptor (NR2A subunit)-deficient mice to see the recovery of morphine analgesia. ICS-treated NR2A-KO mice successfully demonstrated the complete recovery of morphine analgesia, when given intracerebroventricularly (i.c.v.), without any change in the basal nociception threshold. The i.c.v. pre-administration of siRNA for NR2A or NR2A antagonist, (R)-CPP recovered the potency of morphine analgesia under the established pain state. Similar results were also observed with systemic (i.p.) dextromethorphan, which has NMDA receptor antagonist activity. Lastly, we found that the single administration of methadone, which has opioid agonist activity and NMDA receptor antagonist activity, showed potent analgesic actions, as seen in the case with naïve mice.

Central post-stroke pain (CPSP) is one of the secondary diseases of cerebral stroke. However, the detailed mechanism remains unclear. Recently, it is reported that central administration of orexin-A reduces nociceptive responses in inflammatory pain model mouse. In this study, we tested the effect of orexin-A on CPSP model mouse. Male ddY mice (5 weeks old) were subjected to 30 min of bilateral carotid artery occlusion (BCAO). Mechanical allodynia was measured by von Frey filament test. The withdrawal responses to mechanical stimuli were significantly increased on day 3 after BCAO. On day 3 after BCAO, prepro-orexin (orexin precursor) was decreased as compared with sham. The BCAO-induced mechanical allodynia suppressed by the intracerebroventricular (i.c.v.) injection of orexin-A. These effects of orexin-A were inhibited by the intrathecal injection of yohimbine (an adrenaline α₂ receptor antagonist) or WAY100635 (a serotonin 5-HT_1A receptor antagonist). A c-Fos (a neuronal activation marker) expression was observed in the rostral ventromedial medulla or locus coeruleus after i.c.v. injection of orexin-A. These results suggested that orexin-A plays an important role in the regulation of CPSP mediated by the descending pain control system.

Aims; We previously reported that the duration of postsurgical pain was prolonged by the exposure of repeated-social defeat stress (RSDS), and the prolongation was ameliorated by docosahexaenoic acid (DHA). G-protein coupled receptor 40/free fatty acid receptor 1 (GPR40/FFAR1), which is activated by long chain fatty acids, has beneficial effect to pain and emotion. In this study, we examined the involvement of DHA-GPR40/FFAR1 signaling in RSDS induced-chronic pain.

Methods; GPR40/FFAR1 wild type (GPR40^wt) and knockout (GPR40^ko) mice (9 weeks old) were exposed RSDS. Postsurgical pain was induced by plantar incision. Vehicle or DHA (25 mmol/kg) was orally administrated once a day during RSDS. Paw withdrawal thresholds (PWT) were evaluated using by von Frey filaments.

Results; In RSDS-GPR40^wt, vehicle or DHA treated mice showed decrease of PWT on day 1 after paw surgery. However, on day 7 after paw surgery, decrease of PWT disappeared in DHA treated mice but not vehicle treated mice. In RSDS-GPR40^ko, decrease of PWT occurred on day 1 after paw surgery. DHA did not ameliorate decrease of PWT in RSDS-GPR40^ko on day 7 after paw surgery.

Conclusion; DHA-GPR40/FFAR1 signaling may be therapeutic target of chronic pain associated with emotional disorders.

The effect of neonicotinoids on methylation of brain genomic DNA

Neonicotinoids, including dinotefuran and imidacloprid, are agricultural chemicals widely used throughout the world, acting selectively on insect acetylcholine receptors. However, in recent years, it has been shown the possibility that pups' anxiety behaviors may be caused by oral administration of neonicotinoids to pregnant mice, so the influence of neonicotinoids on brain development has been concerned.

　The previous studies have suggested that oral administration of neonicotinoids to the mice affects the methylation of genomic DNA, which is a regulatory mechanism of epigenetic gene expression. However, we have not identified the target genes of methylation yet. In this study, we orally administered imidacloprid and dinotefuran to ICR female mice of 12 week-old and investigated the gene methylation in cerebral cortex 4 h after administration of neonicotinoides. Then, we found increased or decreased methylation of several genes and confirmed that the methylation status of those genes was different in each drug.

　In this presentation, we show the results of gene methylation and the expression profile of several genes, including cux1 and begain, which play important roles in brain development, by using quantitative RCP.

Myocardial infarction (MI) is an ischemic heart disease caused by occlusion of coronary artery. Previous studies have shown that pH in the hearts decreases to 5.5-6.5 after MI. However, physiological significance of pH reduction remains largely unknown. Therefore, we have studied proton-sensing G protein-coupled receptors (proton-sensing GPCRs) which are activated under low pH condition. So far, four proton-sensing GPCRs have been reported. Among them, GPR4 has the highest expression level in mouse heart. Thus, we studied the role of GPR4 in pathological responses after MI, to elucidate the physiological significance of pH reduction.

We found that GPR4 is highly expressed in vascular endothelial cells which express cell adhesion molecules and promote infiltration of leukocytes. Expression level of cell adhesion molecules was significantly suppressed in GPR4 KO mice. Infiltration of neutrophils and expression of inflammatory cytokines were also suppressed. Cardiac function after MI was improved in GPR4 KO mice compared to WT mice.

In this study, we demonstrated that pH decrease after MI activates GPR4 and induces inflammatory responses leading to heart dysfunction. These results indicate that GPR4 can be a new therapeutic target for the treatment of MI.

Purinergic P2Y6 receptor (P2Y6R) is a member of G protein-coupled receptor (GPCR) activated by uridine nucleotides. We previously reported that MRS2578, a specific inhibitor of P2Y6R, suppressed cardiac remodeling and dysfunction induced by pressure overload (EMBO J, 2008). We thus hypothesized that P2Y6R deficient mice would show the same phenotype and performed transverse aortic constriction (TAC) to induce cardiac pressure overload in P2Y6R deficient mice. Contrary to our expectation, P2Y6R deficiency exacerbated cardiac remodeling induced by pressure overload. Surprisingly, transgenic mice with cardiomyocytes-specific overexpression of P2Y6R was also found to worsen cardiac remodeling after pressure overload. We thus speculated that MRS2578 could activate P2Y6R-dependent cardioprotective signaling pathway. We found that MRS2578 upregulated survival factors in mouse hearts, such as SOD2 and SIRT3 in a P2Y6R-dependent manner. We further found that MRS2578 treatment multimerized P2Y6R proteins, which were translocated from plasma membrane to cytosol. These results suggest that MRS2578-induced changes in localization and protein quality of P2Y6R is essential for the activation of cardioprotective signaling pathways.

The heart is capable of adapting to various environmental stresses by flexibly changing its structure. Hemodynamic overload (or mechanical stress) and cardiotoxic drugs, such as doxorubicin, cause pathological cardiac remodeling in which abnormal production of reactive oxygen species (ROS) plays a critical role. We have recently showed that functional coupling between transient receptor potential canonical (TRPC) 3 and NADPH oxidase 2 (Nox2) mediated aberrant ROS production during pathological remodeling of mouse hearts. In this study, we found that extracellular ATP caused atrophy of neonatal rat cardiomyocytes (NRCMs), which was attenuated by silencing TRPC3 or Nox2 genes. ATP increased expression of muscle atrophy-related E3 ubiquitin ligase, MAFbx. TRPC3 and Nox2 were responsible for ATP-induced ROS production and atrophy of NRCMs. We demonstrated that ATP treatment caused physical interaction between TRPC3 with Nox2 in TRPC3 and Nox2-overexpressing HEK293 cells. The ATP-induced increase of TRPC3-Nox2 interaction was also observed in NRCMs as well. In summary, ATP may induce cardiomyocyte atrophy through increases in MAFbx expression and ROS production by activating TRPC3-Nox2 functional coupling.

A superoxide-generating NADPH oxidase (NOX) has been reported to mediate cytotoxicity induced by cigarette smoke components. However, the underlying molecular mechanisms and NOX isoform involved have not been fully clarified. Among NOX isoforms identified so far, NOX1 and NOX4 were expressed in rat H9c2 cardiomyocytes. When H9c2 cells were exposed to acrolein or methyl vinyl ketone (MVK), major toxic components of cigarette smoke extracts, a dose-dependent decline in cell viability was observed. Unexpectedly, disruption of Nox1 significantly exacerbated cytotoxicity induced by acrolein or MVK. The levels of total and reduced glutathione (GSH) were significantly reduced in Nox1-disrupted H9c2 clones. In these clones, expression of multidrug resistance-associated protein 1 (MRP1), which mediates glutathione efflux, was significantly up-regulated. Treatment of reversan, an MRP1 inhibitor, partially but significantly blunted the cytotoxicity of acrolein and MVK in Nox1-disrupted cells. Taken together, NOX1/NADPH oxidase regulates the expression of MRP1 to maintain intracellular GSH levels in cardiomyocytes and protect against cytotoxic components of cigarette smoke extracts.

Oligodendrocytes are responsible for myelin sheath formation in axons and are compromised in demyelinating diseases, but no treatment has been established to promote their regeneration. In this study, we utilized next-generation sequencing data to analyze the effect of miconazole and clobetasol—two inducers of myelination—on the transcriptome in mice. We found that sterol regulatory element binding factor (SREBF) was involved in myelination. We then screened for SREBF-activating compounds in silico and found the PPARα agonist fenofibrate. Subsequently, we created a transgenic zebrafish line that expresses mCitrine fluorescent protein in oligodendrocytes, under myelin basic protein promotor control. We administered fenofibrate to this zebrafish and performed in vivo imaging by high content screening, and found increased fluorescence signal in oligodendrocytes. Moreover, administering gemfibrozil, another PPARα agonist, to the zebrafish also resulted in a similar increase. This increase induced by fenofibrate or gemfibrozil was suppressed by simultaneous administration of the SREBF inhibitor fatostatin, suggesting that fenofibrate and gemfibrozil induced myelination via SREBF. This approach enables a high-throughput search for novel myelination drugs.

OBJECTIVE: To investigate whether the HMGB1 DAMP signaling pathway is involved inresveratrol anti-oxygen glucose deprivation (OGD)-induced microglialinflammatory processes and explore its underlying mechanisms.

METHODS: Cell viability was tested by MTT assay to determine the appropriateresveratrol and EX527 concentration and OGD time, and the cells were dividedinto four groups: Control, OGD+DMSO, OGD+RES and OGD+RES+EX527, ELISA. Rt-PCRand western blot were used to detect inflammatory factors and HMGB1 signalingpathway-related protein changes. WB and immunofluorescence were used todemonstrate the localization of HMGB1 in cells, the acetylation level of HMGB1and the interaction between HMGB1 and Sirt1 were detected by CoIP. Differentgroups BV2 cells were co-cultured with primary mouse neurons, and the release ofHMGB1 was observed and the level of LDH in the supernatant was detected.

RESULTS: We determined that RES (100umol), EX527 (100umol) and OGD3h were theoptimal treatment conditions. RES inhibited the increase of inflammatorymediators and HMGB1 signaling pathway-related proteins and reduce the increaseof HMGB1 level in cell supernatant after OGD, and EX-527 reversed this effect;immunofluorescence indicated that RES can limit HMGB1 in cells, however,different from the change of HMGB1 in the culture medium, there was nosignificant difference in the mRNA level of HMGB1 in each group, suggesting thatthe increase of HMGB1 level in supernatant after hypoxia is mainly due to theincrease of active secretion rather than synthesis. CoIP results showed that thebinding of HMGB1 to deacetylase SIRT1 was decreased and the level of acetylationwas decreased after OGD. RES could increase the interaction between HMGB1 andsirt1 and increase the acetylation level of HMGB1 but EX527 reduced thisinteraction. In the neuron co-culture system, the extracellular release of HMGB1and LDH was increased in the supernatant of the OGD+DMSO group, while thischange in the RES group was attenuated, and the OGD+RES+EX527 group restored theincrease of LDH and HMGB1.

CONCLUSIONS: The activation of downstream signaling pathway by active release ofHMGB1 is partially involved in OGD induced BV2 inflammatory process. Resveratrolreduces inflammation by inhibiting HMGB1 release, a role associated with itsability to activate SIRT1-mediated acetylation of HMGB1.

Alexander Disease (AxD) is a rare neurodegenerative disease which is caused by dominant gain of function mutations in the GFAP gene. In AxD astrocytes show reactive phenotype with Rosenthal fibers which comprise of GFAP accumulations with aB-crystalline. Although AxD is thought to be an astrogliopathy, emerging evidence indicates that the inflammatory response including the microglial activation is induced in AxD. Accordingly, we previously observed the increase in the number of Iba1-positive microglia in the hippocampus from hemizygotes of 60TM AxD model mice which express human GFAP with R239H mutation (60TM mice). To understand the role of microglia in AxD, we depleted microglia from 60TM mice by treatment with PLX5622 (PLX), a CSF-1 receptor antagonist, between P21 and P42. PLX eliminated almost all Iba1-positive signals and its mRNA level in the hippocampus. Surprisingly, PLX increased GFAP staining and Fluoro-Jade B staining, suggesting exacerbation of AxD pathogenesis. PLX also augmented mRNA levels of reactive astrocyte markers such as Vim, C3 and Cd44. These data suggest that microglia may play a beneficial role in AxD pathogenesis by restraining the astrocyte to show reactive phenotype in this AxD model at early-developmental stage.

Progranulin (PGRN), a cysteine-rich secretory protein, is implicated in neuronal protection. In the central nervous system, it has been reported that PGRN protects against neuroinflammation after cerebral ischemia. On the other hand, granulin (GRN), which is cleaved from PGRN by neutrophil elastase, exerts pro-inflammatory effects. However, roles of PGRN and GRN after cerebral ischemia have not been fully determined. In this study, we examined time-course of changes in the levels of PGRN and GRN and their cellular sources after cerebral ischemia. A rat microsphere-embolism (ME) model and primary cultured microglia isolated from cerebral cortex were used. Protein and mRNA levels of PGRN were increased in the ischemic region of cerebral cortex on day 3 after ME. Furthermore, expression of PGRN was increased in activated microglia after ME. Elastase activity in cerebral cortex was increased on day 1 after ME. GRN protein was increased on day 1 after ME. These results suggest that increased elastase activity causes cleavage of PGRN, and then GRN may promote inflammatory responses after ischemia. Thus, the changes in the levels of PGRN and GRN might contribute to pathological alterations in ischemic disorders.

Accumulation of amyloid-β peptides (Aβ) in the brain triggers the onset of Alzheimer's disease (AD). Therefore, promotion of Aβ clearance is a promising strategy for AD therapy. We previously demonstrated that primary-cultured rat microglia phagocytose Aβ, and the transplantation with rat microglia ameliorates the Aβ burden in brains of Aβ-injected rats. In this study, we demonstrated that stimulation with colony-stimulating factor-1 (CSF-1) efficiently differentiated mouse bone marrow (BM) cells into BM-derived microglia-like (BMDML) cells. BMDML cells effectively phagocytosed Aβ in vitro, and it was comparable to that of primary-cultured mouse microglia. We further found that intra-hippocampally injected BMDML cells migrated directionally toward Aβ plaques in a mouse model of AD in comparisons with a simulation assuming a uniform distribution of cells. Finally, we detected Aβ phagocytosis by BMDML cells concomitant with a reduction in the number and area of Aβ plaques and amelioration of the cognitive impairment in the mouse model. Our results suggest that BMDML cells could be used in cell-based disease-modifying therapies against AD.

We have recently demonstrated that phosphorylation of SMAD specific E3 ubiquitin protein ligase 2 (Smurf2) at Thr249 (Smurf2^Thr249) plays an essential role for maintenance of stemness of the mesenchymal stem cells (Iezaki, Hiraiwa et al., Development 2018). Glioblastoma (GBM) is the most common high‐grade malignant glioma in adults. Emerging evidence indicates that glioma-initiating cells (GICs) contribute to drug resistance and tumor recurrence owing to their ability for self-renewal. Here, we show that phosphorylation of Smurf2^Thr249 plays an important role for maintenance of stemness of GICs and GBM progression. Smurf2^Thr249 phosphorylation was markedly decreased in GBM patients as well as in GBM model mice. Infection of Smurf2(T249A) mutant, in which the threonine was replaced with an alanine to prevent phosphorylation, significantly increased not only sphere formation ability of human GICs but tumor progression in a GBM mouse model. On the contrary, Smurf2 phospho-mimetic mutant (Smurf2(T249E)) decreased both of them. These findings highlight a critical role of Smurf2^Thr249 phosphorylation in maintenance of stemness of GICs and GBM tumorigenesis, thereby providing a novel target in GBM.

Hematopoieticstem cells(HSCs) and leukemic stem cells(LSCs) have self-renewal ability to maintain normal hematopoiesis and leukemia propagation, respectively. Recently, it has been reported that bone forming osteoblasts provide a microenvironment for LSCs and are implicated in pathogenesis and progression of leukemia as an osteoblastic niche in bone marrow. The mTOR complex 1 (mTORC1), a member of the serine/threonine kinases, is known to regulate the cellular function in various cell types. Although the role of osteoblastic mTORC1 on bone mass accrual has been investigated, here we show a critical role of mTORC1 in regulating normal hematopoiesis and leukemia progression through its expression in osteoblasts in mice. Using a mouse models of acute myeloid leukemia (AML), we revealed that AML cells enhance the mTORC1 activity in osteoblasts in vivo and in vitro. Subsequent analyses determined that inactivation of Tsc1, a negative regulator of mTORC1, in osteoblasts results in a marked acceleration of AML. These findings highlight a critical role of mTORC1 in normal hematopoiesis and leukemia propagation, at least in part, through its expression in osteoblastic niche.

Smad molecules (Smad1-8) mediate BMP/TGF-β signaling and play important roles in various biological responses such as development and cancer progression. However, the mechanisms regulating stabilities of Smad proteins remain largely unknown. We found that Smad6 protein is more stable than other Smad family proteins, and revealed the molecular mechanisms that regulate Smad6 protein stability.

 We first searched for the Smad6-interacting proteins that can affect Smad6 protein stability. The LC-MS/MS analysis revealed an ubiquitin ligase that directly interacts with Smad6. We found that the ubiquitin ligase polyubiquitinated Smad6, but unexpectedly promoted Smad6 protein stability. The ubiquitin assay using various Smad6 mutants identified the 7 lysine residues of Smad6 that are ubiquitinated by the ubiquitin ligase. Consistently, a Smad6 mutant in which the 7 lysine residues were replaced by arginine (Smad6 KR) did not undergo polyubiquitination by the ubiquitin ligase, and the protein stability of Smad6 KR was much lower than that of Smad6 WT. Smad6 was reported to worsen clinical condition of breast cancer by inhibiting BMP signaling. Interestingly, the tumorigenicity of Smad6 KR was much lower than that of Smad6 WT.

Melanoma is the most malignant form of skin cancer, which originates from the pigment-producing melanocytes in the basal layer of epidermis. L-type amino acid transporter 1 (LAT1) is an amino acid transporter that transports most of the essential amino acids. LAT1 is upregulated in various cancers, including melanoma, and contributes to supporting cancer cell proliferation. Therefore, LAT1 is regarded as a promising molecular target of novel anti-cancer therapeutics. In this study, we established a syngeneic and orthotropic mouse model for melanoma to examine the anti-tumor effects of LAT1 inhibitor. B16F10 mouse melanoma cells were orthotopically injected into footpads of C57BL/6J mice. One week after the inoculation, intravenous injection of LAT1 inhibitor was started and continued once-daily for two weeks. The tumor size on footpads of LAT1 inhibitor-treated mice was significantly reduced compared to those of saline-treated control mice, demonstrating the anti-tumor effects of LAT1 inhibitor. Our syngeneic and orthotropic model will be useful to evaluate the therapeutic efficacy of LAT1 inhibitors for melanoma in the presence of intact immune system, and also to investigate its detailed mechanisms of action as anti-tumor therapeutics.

Alanine-serine-cysteine transporter 2 (ASCT2), a neutral amino acids transporter, plays a central role in sustaining glutamine metabolism. L-type amino acid transporter 1 (LAT1), a large neutral amino acids transporter, transports most of the essential amino acids. Both of them are highly expressed in various type of cancers. In the present study, we investigated the combinational effect of ASCT2 inhibitor and LAT1 inhibitor in four colorectal cancer cell lines in cell proliferation experiments in vitro. Treatment with ASCT2 or LAT1 inhibitor alone showed limited growth inhibition in all the tested cell lines. Treatment with the combination of two inhibitors showed prominent synergistic growth inhibition in two cell lines, additive growth inhibition in one cell line, while there was no obvious combinational effect in the remaining one cell line. LAT1 and ASCT2 expression, transport function, amino acids content and signaling in these cell lines will be further studied, to reveal the cellular determinants underling the difference among cell lines. The results of this study may contribute to propose combinational treatment for colorectal cancers using inhibitors of the two amino acid transporters.

Nafamostat has been reported to cause the drug-induced hyperkalemia. Nafamostat has an inhibitory effect on the serine proteases including tissue kallikrein. The study aimed to examine involvement of tissue kallikrein in the nafamostat-induced hyperkalemia.

Seven-week-old male Wistar-Imamichi rats were used. Nafamostat (6～18mg/kg) or amiloride（3mg/kg）, a potassium-sparing diuretic and a positive control, was i.p. administered. Urine and blood were collected 6 h after administration. Potassium and creatinine (Cr) were measured by the ion-electrode and Jaffe methods, respectively. Tissue kallikrein was measured using the synthetic peptide of the substrate.

In the nafamostat group (n=5), serum potassium and urinary potassium and tissue kallikrein were 5～5.3 mmol/L (control group 4.6±0.2, mean±SD, n=4), 0.33～0.58 mmol/mg Cr (0.56±0.14) and 0.02～0.10μmol/mg Cr (0.11±0.04). In the amiloride group (n=3), they were 6.3±0.6 mmol/L (4.7, mean, n=2), 0.28±0.20 mmol/mg Cr (1.02), 0.06±0.01μmol/mg Cr (0.26), respectively. It was suggested that serum potassium increased and urinary potassium and kallikrein decreased in both groups. In the nafamostat group, redness on the peritonea and hemorrhagic ascites were observed.

Nafamostat-induced hyperkalemia may be involved in the sodium channel inhibition. Intraperitoneal findings may be caused by the anticoagulant effect. Further studies are necessary on the i.v. administration of nafamostat.

A critical role of oxidative stress in the development of nonalcoholic fatty liver disease (NAFLD) has been reported. To clarify the source of oxidative stress, hepatic expression of the superoxide-generating NADPH oxidase isoforms was examined in high-fat and high-cholesterol (HFC) diet-fed mice. The level of NOX1, but not of NOX2 or NOX4 mRNA was significantly elevated in the liver of mice fed HFC diet for 8 weeks. Increased levels of serum alanine aminotransferase and hepatic cleaved caspase-3 in HFC-fed wild-type mice (WT) were significantly ameliorated in mice deficient in Nox1 (Nox1-KO). Formation of nitrotyrosine adducts, a marker of peroxynitrite-induced injury, was apparent in hepatic sinusoids of HFC diet-fed WT, which was significantly suppressed in Nox1-KO. In fact, NOX1 mRNA was predominantly expressed in the fraction of liver sinusoidal endothelial cells (LSECs). In primary cultured LSECs, palmitic acid, the most abundant saturated free fatty acid in plasma, dose-dependently up-regulated NOX1 mRNA. Accordingly, increased oxidative stress mediated by NOX1/NADPH oxidase in LSECs may promote liver injury through peroxinitrite formation in the development of NAFLD.

Muscle atrophy is a result of aging and disease, and can compromise physical function and impair vital metabolic processes. Low levels of muscular fitness, known as ‘flail', contribute greatly to weakness, disability, and immobility, and lead to increased hospitalization and loss of independence.

　To investigate the correlation between muscle atrophy and aging-related immune deterioration, a dissected sciatic nerve sarcopenic mouse model was established and the fraction of immunological cells, including T cells, B cells, macrophages, natural killer cells, and neutrophils, was determined. Eight weeks after sciatic nerve dissection, the volume of both hind legs was evaluated based on acquired magnetic resonance imaging. Primary splenocytes and biceps femoris muscle-derived cells were isolated and labeled by cellular markers. The subpopulation of the cells was then detected using flow cytometry. T and B cell lineages were significantly suppressed in the sarcopenic model compared with control mice, and the fraction of macrophages and natural killer cells also tended to increase.

　In conclusion, muscular loss appears to affect the immunological system by modulating humoral immunity and the cell-based immunology response. Further functional analysis of individual cell subsets could further determine the influence of sarcopenia on immune system improvement.

Patch-clamp recordings are useful to investigate the electrophysiological dynamics of neurons at single synapse level with high time resolution. However, subthreshold membrane potentials (Vm) recorded from in-vivo animals include complex synaptic inputs from thousands of presynaptic neurons, and it is technically difficult to remove artifacts induced by respiration and blood vessel pulsation. The goal of this research is to quantify the amplitude of a synaptic input in in-vivo synaptic bombardments using deep learning, which is designed to recognize natural images with high accuracy. We recorded spontaneous Vm fluctuations from hippocampal CA1 pyramidal cells in anesthetized mice and a pseudo-ideal excitatory post synaptic potential (EPSP) from a CA1 pyramidal cell in a hippocampal slice. The in-vivo Vm fluctuations were randomly phase-shifted to distort the waveform of intrinsic synaptic inputs and were superimposed with a single EPSP with various amplitudes, then we yielded surrogate Vm images with and without EPSPs. We trained ResNet, a deep learning model, with this dataset to estimate the amplitudes of EPSPs embedded in Vm fluctuations. We succeeded in reducing the mean error of the prediction to a level of the standard deviation of spontaneous Vm fluctuations.

Background: Oxaliplatin causes oxaliplatin-induced peripheral neuropathy (OIPN). Because OIPN reduces patients' quality of life (QOL), the development of preventive agents against OIPN is an important area of research. In this study, we searched for preventive drugs against OIPN by drug repositioning, using large-scale medical data, and experimentally validated the findings with neuron-like cells and OIPN rat models.

Methods and Results: First, we searched for approved drugs that cancel the gene expression change caused by oxaliplatin, using the drug discovery tool LINCS, and found 23 approved drugs that met the requirements. Candidate drugs were then evaluated for their mitigating effects on OIPN, using the FDA Adverse Event Reporting System (FAERS) database, and Drug X was found to significantly reduce the risk of patients developing OIPN. Using PC12 cells, we observed significant improvement in oxaliplatin-associated axonal damage with Drug X treatment. In addition, in vivo experiments showed that Drug X significantly reduced the expression of oxaliplatin-induced neuropathy in OIPN rat models.

Conclusion: This study suggested that drug repositioning of Drug X could allow it to be used as a preventive agent for OIPN.

In multiple sclerosis (MS) patients, pain is a frequent and disabling symptom. However, the underlying mechanisms of neuropathic pain in MS patients is poorly understood. In the present study, we have demonstrated that cathepsin E (CatE) in neutrophils is required for mechanical allodynia in experimental autoimmune encephalomyelitis, an animal model of MS. We show that CatE-deficient (CatE -/- ) mice were highly resistant to myelin oligodendrocyte glycoprotein (MOG 35- 55 )-induced mechanical allodynia. After MOG 35-55 immunization, neutrophils immediately accumulated in the dorsal root ganglion (DRG) where neutrophils released elastase in a CatE- dependent manner. Furthermore, sivelestat, a selective neutrophil elastase inhibitor, suppressed mechanical allodynia caused by adoptively transferred MOG 35-55 -stimulated neutrophils. Neutrophil- driven increased pain perception was mediated through the activation of protease-activated receptor 2 in DRG neurons. Activation of neutrophils by MOG 35-55 was mediated by toll-like receptor 4. Our findings suggest the mechanism of driving mechanical allodynia caused by MOG 35-55 and new strategy for preventing pain in MS patients.

Orexin, a neuropeptide produced by neurons in the lateral hypothalamus, regulates sleep and wakefulness. Orexin deficiency causes narcolepsy-cataplexy characterized by excessive sleepiness and cataplexy, a sudden loss of muscle tone triggered by emotion.

We previously showed that peripherally (i.p.) administered YNT-185 (40 mg/kg), an OX2R-selective agonist, ameliorates narcolepsy-cataplexy symptoms in mouse models. However, repeated i.p. of YNT185 can be stressful and the effective dose for oral administration (p.o.) is too high (4000 mg/kg) to ameliorate the symptoms. Here we further optimized YNT-185 (EC₅₀ ≈ 28 nM) and produced YNT-X, which increased intracellular Ca²⁺ in lower concentrations in OX2R-transfected cells. The EC₅₀ value was 1.1 nM (OX2R) and the maximum response was similar to orexin in vitro. The response was inhibited by EMPA, an OX2R antagonist. In vivo, YNT-X p.o. increased wake time in wildtype mice in a dose-dependent manner, with the effective dose of 2.5-10 mg/kg, which is several hundred times lower than YNT-185. Our results suggest that YNT-X may be useful as mechanistic, oral therapy for narcolepsy-cataplexy.

Characterization of cells in the ventral tegmental area activated by abused drugs

Generally, activation of the mesolimbic dopaminergic system, which projects from the ventral tegmental area (VTA) to the nucleus accumbens (N.Acc.), plays an important role in the development of psychological dependence on drugs of abuse. Medium spiny neurons in the N.Acc., which include dopamine D₁ receptors and mostly project to the VTA, are likely to be associated with relapse in drug abuse. Abused drugs have been classified as "uppers" and "downers," and it is believed that they cause drug addiction via different networks. In this study, we characterized cells in the VTA that are activated by abused drugs. In cFos-EGFP-Rp110a (cFos-TRAP) mice, drug-activated cells and non-activated cells were analyzed by FACS. As a result, most of the neurons in the VTA that were activated by the administration of either morphine or EtOH expressed tyrosine hydroxylase, dopamine transporter and mTOR. These results suggest that abused drugs may selectively activate dopaminergic neurons containing mTOR in the VTA. We are currently trying to identify the neurons that are activated by methylphenidate, benzodiazepines and other abused drugs to elucidate a common mechanism of drug addiction. This approach may help us understand the complex mechanism of psychological dependence on abused drugs.

Dopamine (DA) is necessary for motor function, motivation, working memory, and reward. However, how DA regulates reward-related learning and memorythrough the gene expression is not fully understood. Neuronal Per Arnt Sim domain protein 4 (Npas4), a brain-specific basic helix-loop-helix transcription factor, plays a role in synaptic plasticity by regulating the expression of activity-dependent genes,such as BDNF.Although Npas4 required forcontextual fear memory formation in mice, the role of Npas4 in reward-relatedlearning and memoryremains unknown. To this purpose, we used the conditioned place preference (CPP) paradigm in which animals learn to prefer a context associated with cocaine. Here, we found that the deletion of Npas4 in the accumbal D1R-expressing MSNs (D1R-MSNs) suppressedCPP. This phonotype was rescued by the D1R-MSNs specific expression of Npas4-WT but not phospho-deficient Npas4 mutant. These results suggestthat Npas4 and its phosphorylation regulate reward-related learning and memory in the accumbal D1R-MSNs.

Amyotrophic lateral sclerosis (ALS) is a motor neuron-specific neurodegenerative disease and frontotemporal dementia (FTD) is a neurodegenerative disease with young-onset dementia. Accumulating evidence indicates that they have common clinical and pathologic features. Several mutations of the CHCHD10 (C10) gene have been found to cause ALS/FTD. Wild-type C10 is localized at mitochondria and physiologically involved in the regulation of mitochondrial function. It has been previously shown that a mutant C10 induces mitochondrial dysfunction such as the reduction in ATPase production that has been hypothesized to be closely linked to the ALS/FTD onset. In the present study, we have investigated the molecular mechanism underlying neuronal cell death caused by a mutant C10, S59L-C10. We have found that the adenovirus-medicated overexpression of wild-type C10 and S59L-C10 induces cell death in vitro. As expected, S59L-C10-induced cell death was more prominent than that induced by wild-type C10. This result suggests that S59L-C10-induced cell death may be caused by the gain-of-toxic mechanism. We have further characterized the pathway in detail underlying the S59L-C10-induced cell death.

[Background] Aggregation of α-synuclein (αS) is promoted by polyunsaturated fatty acids such as arachidonic acid (AA). Fatty acid-binding protein 3 (FABP3) is crucial for AA transport in the brain. We previously reported that FABP3 aggravates AA-induced αS oligomerization and cell death (Shioda et al, J Biol Chem, 2014). We here developed novel FABP3 ligands, MF series, and addressed whether these ligands could suppress αS pathology and Parkinsonism in mice.

[Methods] Mice treated with 1-methyl-1,2,3,6-tetrahydropiridine (MPTP) were chronically administrated with MF1 (high affinity for FABP3), MF4 (low affinity for FABP3), or L-DOPA.

[Results] MPTP-induced motor deficits were ameliorated by MF1 like L-DOPA. On the other hand, αS accumulation in dopaminergic (DA) cell bodies and neuronal loss in the substantia nigra (SN) of MPTP-treated mice were ameliorated by MF1, but not by L-DOPA nor MF4. Finally, MF1 also inhibited αS oligomerization and hyper-phosphorylation in the SN/VTA of MPTP-treated mice.

[Conclusion] MF1 but not L-DOPA suppressed αS pathology, thereby rescuing MPTP-induced DA neuronal loss and Parkinsonism. We propose novel FABP3 ligands as a candidate for synucleinopathies.

ATP-sensitive potassium channel (KATP) is a kind of inwardly rectifying potassium channel that suppresses depolarization in both cell membrane and mitochondria, and has an important role to make and keep resting membrane potential. It is known that activation of KATP protected the cells from ischemic damage in heart and brain. However, the underlying mechanism is not clear. Here, we have investigated that how minoxidil suppresses ischemic damage and excitotoxicity. Transient ischemia model mice were prepared by 1-h middle cerebral artery occlusion using 6-week old male C57/BL mice. Injection of minoxidil immediately after the operation prevented the damage in a concentration-dependent manner. N-methyl-D-aspartic acid (NMDA) induced mitochondrial depolarization after the increase in calcium influx into the cells. Increasing mitochondrial depolarization correlated to extent of neuronal degeneration, while pre-treatment of minoxidil inhibited it. Therefore, these results suggested that the decrease in intracellular potassium level may suppress the neurodegeneration via suppression of mitochondrial depolarization.

DNA methylation of promoter region is thought to be involved in pathogenesis of several diseases. In cerebral ischemia, some studies have reported that infarct volume is decreased by inhibition of DNA methyltransferases (DNMTs) that mediates DNA methylation. However, roles of DNMTs in cerebral ischemia have not been clarified. Therefore, we investigated whether DNA methylation was associated with pathology of cerebral ischemia.

In this study, an in vivo cerebral ischemia was produced by occlusion of middle cerebral artery and reperfusion (MCAO/R) in the rat. First, we investigated protein levels of DNMTs. Protein levels of DNMT3a, but not DNMT3b, were increased in penumbra regions 1 day after MCAO/R compared with those in same regions of sham-operated rats. Also, Immunohistochemical examination 1 day after MCAO/R revealed that DNMT3a-positive cells in penumbra regions were colocalized with NeuN-positive neurons. Therefore, we determined effect of DNMTs inhibitor RG108 against N-methyl-D-aspartate (NMDA)-induced neuronal cell death in primary cultured rat cortical neurons. RG108 protected neurons from NMDA-induced cell death.

These results suggested that neuronal cell death after cerebral ischemia was correlated with DNA methylation by DNMT3a.

Ischemic neuronal damage is induced by various factors such as glutamate excitotoxicity and oxidative stress. Excessive amount of extracellular glutamate followed by cerebral ischemia is a major factor to lead intracellular Ca²⁺ overload through the N-methyl-D-aspartate (NMDA) receptor and then causes neuronal death. It has been known that several proteolytic enzymes are stimulated by intracellular Ca²⁺ elevation. However, roles of proteolytic enzymes on NMDA-induced neuronal damage are not fully defined. We examined whether inhibitions of proteolytic enzymes protected primary cultured rat cortical neurons from NMDA-induced damage. Among several inhibitors, furin inhibitor markedly protected neurons from NMDA-induced damage in a dose-dependent manner. Furthermore, calpain activation led by NMDA treatment was inhibited by the furin inhibitor. We next investigated the ability of furin inhibitor to attenuate cell damage in a rat cerebral ischemia model. We demonstrated that infarct volume after cerebral ischemia was not affected by treatment with furin inhibitor. Although further research will be needed to assess the therapeutic potential of furin inhibitor in cerebral ischemia in vivo, this study revealed a novel role of furin in excitotoxic injury in cortical neurons.

Introduction: Lithium has been widely used for the treatment of bipolar disorder. However, several cardiac adverse events have been noticed in patients who were also treated with other drugs in addition to lithium. In the present study, we studied the effects of lithium by itself to precisely analyze the onset mechanisms of its cardiovascular adverse events.

Methods: We intravenously administered lithium carbonate in doses of 0.1, 1 and 10 mg/kg/10 min to the halothane-anesthetized dogs (n=4) under the monitoring of cardiohemodynamic and electrophysiological variables.

Results: The currently used doses of lithium provided plasma concentrations ranging from sub-therapeutic to toxic ones. The low and middle doses significantly prolonged the ventricular effective refractory period. Additionally, the high dose significantly decreased the heart rate, delayed the intraventricular conduction and the ventricular repolarization, and kept the effective refractory period prolonged.

Conclusion: Lithium may have a wide safety margin against hemodynamic adverse events excpt that it can moderately inhibit both Na⁺ and K⁺ channels, leading to increase of the ventricular refractoriness and decrease of the heart rate.

Human-induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are significantly immature compared with adult cardiomyocytes and exhibit a fetal-like phenotype. Although efficient propagation of electrical signals in the heart is crucial for natural development program and functionality, maturation of hiPS-CMs causes hyperpolarization of resting membrane potentials and a loss of automaticity in vitro. We therefore investigated whether artificial electrical field stimulations mature physiological properties of hiPS-CMs, especially on intracellular sodium concentrations ([Na⁺]_i). We cultured hiPS-CMs for a week under electrical field stimulations (3 V/cm, 1 ms, 1 Hz), and quantified [Na⁺]_i with a Na⁺ indicator. Basal　intracellular sodium concentration without stimuli was 4.7 mM (n = 17) which is much lower than that in normal excitable cells (10-15 mM). The intracellular sodium concentration with the electrical stimuli (5.7 mM, n = 23) was significantly greater than [Na⁺]_i without stimuli. These results suggest that artificial electrical stimulations enable us to obtain more mature hiPS-CMs in vitro.

Inhibition of K⁺-conductance through the hERG channel leads QT prolongation and associates with cardiac arrhythmias. We have found that estrogens interact with a drug-binding site, F656 of the hERG channel, and alter effects of a hERG blocker. However precise mechanistic insights have not been elucidated. We here investigated actions of ethynylestradiol (EE2), a synthetic estrogen, on the hERG channel. HEK293 cells stably-expressing the hERG channels were cultured in the steroid-free medium. The patch-clamp technique is performed to record hERG currents. Supratherapeutic concentrations of EE2 did not alter amplitudes and kinetics of the hERG currents elicited with train pulses at 20 mV (0.1 Hz). On the other hand, EE2 recovered the hERG inhibition induced by E-4031. These results suggest that EE2 interacts with E-4031 at the promiscuous drug-binding site of the hERG channel and imply that EE2, an anti-breast cancer drug or an oral contraceptive, is protective against drug-induced QT prolongation.

The present study investigated the role of p11 in NAc CINs in dopamine responses to rewarding stimuli (cocaine, palatable food and female mouse encounter). The extracellular dopamine and acetylcholine (ACh) levels in the NAc were determined in freely moving male mice using in vivo microdialysis.

Rewarding stimuli induced an increase in dopamine efflux in the NAc of wild-type mice. The dopamine responses were attenuated in constitutive p11 KO mice. The dopamine response to cocaine was accompanied by an increase in ACh NAc efflux, whereas the attenuated dopamine response to cocaine in p11 KO mice was restored by pharmacological activation of ACh receptors in the NAc.

Dopamine response to cocaine and an increase in ACh were attenuated in mice with deletion of p11 from cholinergic neurons (ChAT-p11 cKO mice), whereas gene delivery of p11 to CINs and chemogenetic activation of CINs restored the dopamine responses.

Thus, p11 in NAc CINs plays a critical role in activating these neurons to mediate dopamine responses to rewarding stimuli. The dysregulation of mesolimbic dopamine system by dysfunction of p11 in NAc CINs may be involved in pathogenesis of depressive states.

We analyzed the roles of GABA_A and GABA_B receptors (-Rs) in regulating acetylcholine (ACh) efflux in the nucleus accumbens (NAc) of freely moving rats using in vivo microdialysis. The effects of GABA-R ligands on accumbal dopamine (DA) efflux were also analyzed as accumbal cholinergic and dopaminergic neurons could mutually interact. Drugs used were infused directly into NAc. The GABA_A-R agonist muscimol (30 pmol) and the GABA_B-R agonist baclofen (300 pmol) each reduced accumbal ACh. The GABA_A-R antagonist bicuculline (60 pmol) counteracted the muscimol (30 pmol)-induced decrease in ACh and the GABA_B-R antagonist saclofen (12 nmol) counteracted the baclofen (300 pmol)-induced decrease in ACh. Each of muscimol (30 pmol), baclofen (300 pmol), bicuculline (60 pmol) and saclofen (12 nmol) did not alter baseline accumbal DA when given alone. Doses of compounds indicate total amount infused (mol) during 30-60 min infusions. These results show that GABA_A and GABA_B-Rs exert inhibitory roles in the control of accumbal ACh efflux. This study provides in vivo evidence that GABA_A and GABA_B-Rs each reduce accumbal cholinergic activity without affecting accumbal dopaminergic activity.

Essential tremor (ET) is one of the most common movement disorders, exhibiting a postural and/or kinetic tremor. We previously analyzed tremorgenic gene components using a ET model, Tremor rats, and showed that deletion of aspartoacylase (Aspa) and a missense mutation of Hcn1 channels are implicated in induction of tremor (Exp. Anim. 65, 293–301, 2016). Since monoamines are known to be involved in regulation of tremor induction, here, we evaluated the changes in brain monoamine levels in Aspa-knockout (KO) rats. Dopamine, 5-HT and their metabolites were analyzed in 9 brain regions (cerebral cortex, hippocampus, striatum, thalamus, hypothalamus, midbrain, inferior olive, pons and cerebellum), using a HPLC-ECD method. Aspa-KO rats showed increases in 5-HT levels in the hypothalamus and inferior olive, and in 5-HIAA and 5-HIAA/5-HT levels, in most brain regions examined, indicating that deletion of Aspa gene enhances the 5-HT turnover rates. Alterations in HVA levels were also found some regions (increases: cortex and striatum, decreases: hippocampus and hypothalamus). The present results suggest that enhancement 5-HT synthesis and metabolism, especially in the inferior olive, may be linked to tremor induction.

Cholinergic neurons in basal forebrain (BF) project to various brain regions including cortex and hippocampus. BF receives various inputs such as serotonergic fibers from the dorsal raphe nuclei. However, serotonin (5-HT)-induced modulatory effects on the excitatory synaptic transmission in BF are unknown. This study was aimed to elucidate 5-HT-induced modulation of glutamatergic synaptic transmission onto BF cholinergic neurons. BF cholinergic neurons were identified with Cy3-192IgG and investigated in P12-20 rat brain slices. Excitatory postsynaptic currents (EPSCs) were evoked by focal electrical stimulation. 5-HT, a 5-HT_1A receptor agonist or a 5-HT_1B receptor agonist inhibited the amplitude of EPSCs. In the presence of both 5-HT_1A and 5-HT_1B receptor antagonists, most of 5-HT-induced effect disappeared. 5-HT-induced inhibition was significantly smaller in the presence of ω-agatoxin TK than that without ω-agatoxin TK. 5-HT reduced synaptic strength by changing AMPA/ NMDA ratio. These results suggest that 5-HT inhibits glutamatergic transmission onto BF cholinergic neurons via both 5-HT_1A and 5-HT_1B receptors. They also suggest that 5-HT inhibits glutamatergic transmission by selectively blocking CaV2.1 (P/Q-type).

Histamine acts as a neurotransmitter in the brain. Histamine is synthesized from histidine by catalyzing histidine decarboxylase (HDC). In the central nervous system, HDC-positive neurons are in tuberomammillary nucleus of posterior hypothalamus and project their axons to entire brain. However, the roles of HDC in brain functions are not fully elucidated. In the present study, we generated mice with brain-specific deletion of HDC to investigate the importance of histamine for adult brain.

We stereotaxically microinjected adeno-associated virus (AAV) expressing

Cre-recombinase into tuberomammillary nucleus of adult HDC flox mice (HDC cKO mice). Immunohistochemical analysis showed Cre expression in tuberomammillary nucleus in HDC cKO mice. We confirmed the reduced HDC mRNA expression and the decreased histamine content in HDC cKO brain. Light/dark box tests showed that HDC cKO mice spent a shorter amount of time in the light room. In the tail suspension tests, immobility time was prolonged in HDC cKO mice. These results indicated that the inhibition of HDC activity in adult brain reduced histamine content and induced anxiety- and depression-like behaviors in mice.