[Background]In salt-sensitive hypertension, reactive oxygen species (ROS) play a major role in the progression of renal disease partly via activation of the mineralocorticoid receptor. Previously, we demonstrated that urinary vanin-1 is an early biomarker of oxidative renal tubular injury. However, it remains unknown whether urinary vanin-1 might reflect the treatment effect.

[Objective]This study aimed to clarify the treatment effect for renal tubular damage in Dahl salt-sensitive (DS) rats.

[Methods]DS rats (6 weeks old) were given one of the following for 4 weeks: high-salt diet (8% NaCl), high-salt diet plus superoxide dismutase mimetic, tempol (3 mmol/L in drinking water), high-salt diet plus eplerenone (100 mg/kg/day), and normal-salt diet (0.3% NaCl). After 4 weeks of the treatment, blood pressure was measured by the tail-cuff method and kidney tissues were evaluated. ROS were assessed by measurements of malondialdehyde and by immunostaining for 4-hydroxy-2-nonenal.

[Results]A high-salt intake for 4 weeks caused ROS and histological renal tubular damages in DS rats, both of which were suppressed by tempol and eplerenone. Proteinuria and urinary N-acetyl-β-D-glucosaminidase exhibited a significant decrease in DS rats receiving a high-salt diet plus eplerenone, but not tempol. In contrast, urinary vanin-1 significantly decreased in DS rats receiving a high-salt diet plus eplerenone as well as tempol. Consistent with these findings, immunohistochemical analysis revealed that vanin-1 was localized in the renal proximal tubules but not the glomeruli in DS rats receiving a high-salt diet, with the strength attenuated by tempol or eplerenone treatment.

[Conclusion]These results suggest that urinary vanin-1 is a potentially sensitive biomarker for ameliorating renal tubular damage in salt-sensitive hypertension.

Aim: Erythropoietin (EPO) is essential for the hematopoiesis. Kidney is the major organ to produce EPO in adults, so that kidney damage reduces its level in the body, resulting in anemia. Hypoxia-inducible factor is a well known translational factor to induce adaptive responses in the cell, and plays significant role on the EPO gene translation in the EPO producing cells. Therefore, the inhibitors of prolyl hydroxylase domain-containing protein (PHD) is awaited for one of the new therapeutic strategies against renal anemia. Likewise, it can be predictable that tons of patients who receive the PHD inhibitor have renal dysfunction as a cause of anemia, while the potential reno-protective effects of PHD inhibitor have not been evaluated well. In the present study, we investigated the effects of modulistat, a PHD inhibitor, on anemia and renal dysfunction when initiated after the onset of adenine-induced nephropathy.

Methods: Male C57Bl/6J mice received adenine orally for the induction of nephropathy. After the onset of nephropathy, the mice were separated into 2 groups and received either vehicle or modulistat (3 mg/kg/day, p.o.) for 4 weeks.

Results: There was no difference in hematocrit level between vehicle- and modulistat-treated groups (34.8±0.9 vs. 34.8±0.6%). At 4 weeks of the administration, vehicle-treated mice showed significant anemia (hematocrit level: 28.5±1.0%), and modulistat ameliorated the anemia (44.3±1.4%). Vehicle-treated mice exhibited reduction of creatinine clearance and body weight, increase in blood urea nitrogen, progressed histopathological changes, immune cells infiltration and dehydration. Modulistat improved immune cells infiltration and dehydration but not the other parameters.

Conclusion: Modulistat treatment after the onset of nephropathy could recover mice from anemia. Modulistat improved some parameters of renal abnormality but unable to restore the renal function.

Background: Ferroptosis has been identified as iron-dependent regulated cell death, and it participates with a various disorder including kidney disease. Cisplatin, a classical anti-cancer drug, causes nephrotoxicity, which is inhibited by iron chelator. In the present study, we examined the involvement of ferroptosis on cisplatin-induced nephrotoxicity.

Methods: We used male mice with cisplatin-induced nephrotoxicity that were pretreated with vehicle or a ferroptosis inhibitor. Mice were sacrificed at 48 hours later.

Results: Cisplatin administration actually augmented ferrous iron and hydroxyl radical production in the kidney. Cisplatin-induced COX-2 expression, as well as lipid peroxide, was increased by cisplatin-treated kidney. An inhibitor of ferroptosis also suppressed cisplatin-induced increased of COX-2 expression and lipid peroxide. Mice with cisplatin administration developed kidney injury with renal dysfunction, and showed augmented oxidative stress, increased apoptosis, and elevated inflammatory cytokines. However, most of these symptoms were suppressed by a ferroptosis inhibitor.

Conclusion: Ferroptosis is suggested to involve cisplatin-induced nephrotoxicity.

Emerging evidences provide a microbial imbalance (dysbiosis) is linked to several diseases including cardiovascular diseases. It has been reported that Gram-positive Streptococcus genus is increased in feces of spontaneous hypertensive rat (SHR) with increased intestinal permeability. However, the mechanism by which the dysbiosis induces increased blood pressure remains unknown. We hypothesized that bacterial toxin derived from gut Streptococcus genus in hypertension may cause dysregulation of blood pressure. In this study, we examined the effect of streptolysin O, a streptococcal pyrogenic exotoxin, on vascular function using Wistar rat. Treatment with streptolysin O (10-1000 ng/ml, 30 min) did not change contractile responses to phenylephrine or serotonin in aorta (n=4). In contrast, streptolysin O significantly impaired acetylcholine-induced endothelial dependent relaxation in a dose-dependent manner (n=6, p<0.05), while sodium nitroprusside-induced endothelial independent relaxation was unchanged (n=4). Streptolysin O increased the level of eNOS phosphorylation at Thr495 (n=6). Endothelial dysfunction caused by streptolysin O was attenuated by pan protein kinase C (PKC) inhibitor (Ro 31-8222, n=6, p<0.05), PKCβ inhibitor (LY 333531, n=5, p<0.05) or selective PKCβ2 inhibitor (GCP53353, n=3, p<0.05). We conclude that streptolysin O may cause increment of blood pressure through vascular endothelial dysfunction, which is mediated by PKCβ2-induced phosphorylation of eNOS inhibitory site.

Chemerin is an adipocytokine which binds to several receptors such as chemokine-like receptor 1 (CMKLR1) and chemokine (CC motif) receptor-like 2 (CCRL2). Chemerin-9, an active fragment of chemerin, induces vasoconstriction via CMKLR1. Pulmonary artery hypertension (PAH) is a fatal disease caused by the increased PA resistance. We examined the effects of chemerin-9 on contractility of PA from monocrotaline (MCT)-induced PAH rat. Isometric contraction of isolated PA from MCT-injected (MCT) rat was measured. Protein expression in lung or plasma was measured by Western blotting. CMKLR1 localization in lung was measured by immunostaining. Chemerin-9-induced contraction was significantly enhanced in PA from MCT rat compared with vehicle-injected control (Cont) rat. The CMKLR1 expression was increased, while the expression of CCLR2, a decoy receptor was decreased in lung from MCT rat. The plasma chemerin was increased in MCT rat. CMKLR1 was localized in endothelium of PA from Cont rat, while it was localized in smooth muscle of PA from MCT rat. We for the first time revealed that chemerin-9-induced contraction is enhanced in PA of MCT rat perhaps via increasing CMKLR1 but decreasing CCLR2 in smooth muscle.

Hypertensive disorders of pregnancy (HDP) is a pathological condition with hypertension and vascular endothelial cell dysfunction (inflammation) during the gestational period. Recently, it was reported that reduction level of plasma histidine-rich glycoprotein (HRG) in human pregnant patients was correlated with the HDP seriousness. HRG is an anti-inflammatory factor that controls the progression of systemic inflammatory pathology and mainly produced from liver. However, HRG functions in HDP pathology and perinatal physiology have been still unknown. In this study, we examined the involvement of plasma HRG on placental formation and hypertensive event during the gestational period using C57BL/6 mice and HRG gene-deficient (HRG KO) mice. Plasma HRG level was decreased during the gestational period in C57BL/6 mice. In addition, HRG reduction was restored at post-partum and did not depend on the HRG gene expression in liver. Hysterectomy to the gestational C57BL/6 mice also restored the plasma HRG level up to the normal level. When Human plasma-purified HRG was injected to C57BL/6 mice on nulliparity or gestation, biological half-time of the injected HRG was short in the gestational mice compared with the nulliparous mice. Moreover, HRG KO mice showed hypertension by the gestation, whereas rise in blood pressure did not observed in the gestational C57BL/6 mice. The gestational HRG KO mice had fetal growth disorder and placental hyperplasia. These results suggest that HRG may have a physiological function in the gestational period. Pregnant HRG KO mice may be a HDP-like pathological model that causes abnormalities in the placental formation mechanism controlled by HRG.

[Purpose] Blood pressure (BP) is controlled by the cardiovascular and neuroendocrine system by a complicated manner, in which oxidative stress plays important roles. In this study, roles of Nox2 and Nox4, which are two major subtypes of NADPH oxidases in BP regulation and cerebrovascular events were evaluated using Nox2- and Nox4-deleted stroke prone spontaneously hypertension rat (SHRSP). [Methods and Results] Nox2- and Nox4-deficient SHRSP (Nox2KO and Nox4KO, respectively) were produced by the genome editing technology using the CRISPR/Cas9 system. Body weight of Nox4KO was greater than that of SHRSP at 12 weeks old or later. BP at 12 weeks was elevated in Nox4KO while reduced in Nox2KO when compared with SHRSP. Stroke latency under salt-loading with 1% salt solution was longer in Nox2KO than in SHRSP. [Conclusion] Genetic deletion of Nox2 and Nox4 affected BP and stroke latency in SHRSP. However, as the effects were rather small, Nox2 and 4 might not be major contributors to the development of cardiovascular diseases in SHRSP.

α-Synuclein is an abundant neuronal protein that accumulates in insoluble inclusions in Parkinson's disease and other synucleinopathies. Fatty acids partially regulate α-Synuclein accumulation, and mesencephalic dopaminergic neurons highly express fatty acid-binding protein 3 (FABP3). We previously demonstrated that FABP3 knockout mice show decreased α-Synuclein oligomerization and neuronal degeneration of tyrosine hydroxylase (TH)-positive neurons in vivo. In this study, we newly investigated the importance of FABP3 in α-Synuclein uptake, 1-methyl-4-phenylpyridinium (MPP⁺)-induced axodendritic retraction, and mitochondrial dysfunction. To disclose the issues, we employed cultured mesencephalic neurons derived from wild type or FABP3^-/- C57BL6 mice and performed immunocytochemical analysis. We demonstrated that TH⁺ neurons from FABP3^+/+ mice take up α-Synuclein monomers while FABP3^-/- TH⁺ neurons do not. The formation of filamentous α-Synuclein inclusions following treatment with MPP⁺ was observed only in FABP3^+/+, and not in FABP3^-/- neurons. Notably, detailed morphological analysis revealed that FABP^-/- neurons did not exhibit MPP⁺-induced axodendritic retraction. Moreover, FABP3 was also critical for MPP⁺-induced reduction of mitochondrial activity and the production of reactive oxygen species. These data indicate that FABP3 is critical for α-Synuclein uptake and mitochondrial functions in dopaminergic neurons, thereby preventing synucleinopathies, including Parkinson's disease.

[Background]

Recently, Amyloid β (Aβ) independent pathology is focused attention in Alzheimer's disease (AD) pathology. Among them, endocytic dysfunction is the early pathogenic event before Aβ aggregates. A body of evidence suggested that one of Aβ precursor protein (APP) metabolites, β-carboxyl-terminus fragment （βCTF）, accumulated in endosomes and impaired the endocytic trafficking in AD brain. However, the molecular mechanism is largely unknown. Previously, we identified TMEM30A (CDC50A) as the candidate partner for βCTF toxicity. TMEM30A is a subcomponent of lipid flippase which translocate phospholipids such as a phosphatidylserine (PS) from outer to inner side of lipid bilayers. In this study, we aimed to analyze how TMEM30A and βCTF complex associates with vesicular trafficking in AD.

[Methods]

To investigate the molecular mechanism of βCTF mediated traffic impairment, we established BACE1 (β-secretase) stable expression in SH-SY5Y cells. We analyzed the event induced by complex formation of TMEM30A and βCTF using biochemical approach.

[Results]

We found that βCTF accumulation caused the interaction between TMEM30A and βCTF, which resulted in endosomal characteristic change in SH-SY5Y(BACE1). Moreover, this complex formation affected on PS localization in endosomes, which indicated the alteration of lipid flippase activity.

[Conclusion]

Our study suggests that TMEM30A and βCTF complex can induce traffic impairment via lipid flippase dysfunction in AD. Although further analysis is required, our findings may contribute to the development of a novel AD therapy based on the improvement of vesicular traffics.

The onset and progression of Alzheimer's disease (AD) correlate with neuroinflammatory processes, and inflammatory microglia (MG) are associated with AD-like pathology in a transgenic mouse model. We recently developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG, and identified the antigen molecule for 9F5: truncated form of rat GPNMB/osteoactivin (Kawahara et al., GLIA, 2016). However, the distribution and function of GPNMB+ type 1 MG in AD brain are largely unknown. In the present study, we observed GPNMB+Iba1+ MG surrounding Aβ plaque in neocortex of amyloid precursor protein (APP23) transgenic mice. In addition, GPNMB+Iba1+ MG were observed in non-plaque areas of hippocampus of APP23 mice. We generated Gpnmb knockout mice to investigate the functional relevance of GPNMB for microglia in vivo. Homozygous Gpnmb-KO mice did not show any growth retardation including body weight loss, and the fertility was normal. We observed that AD-related brain dysfunction in APP23 and 5xFAD mice were regulated by Gpnmb gene dosage. These finding suggest that GPNMB-positive type 1 MG may play a role in regulation of neuropathological process of AD.

Alzheimer's disease (AD) is a complex, incurable, and fatal neurodegenerative disorder characterized by progressive memory loss with a very high mortality rate. AD neuropathological alterations begin years before the onset of clinical symptoms and about 99.6% of candidate drugs targeting Aβ plaque the causal factor of AD have failed at various stages of clinical trials. Thus, early diagnosis is an essential requirement for effective intervention. Phosphorylation of collapsin response mediator protein 2 (CRMP2), a member of the CRMPs family, at the S522 site has been previously reported to be aberrantly high in end-stage AD. In this study, the key phosphosignaling events at the early stage of AD were investigated using hippocampal phosphoproteomic analysis of the App^NL-F mice model of early-stage AD. This was followed by protein class enrichment and pathway analyses of the overrepresented protein classes and pathways in the upregulated and downregulated phosphosites. Validation of phosphosites of the AD-related proteins identified by the enrichment analyses of the early-stage AD mice hippocampus was carried out using the medial-temporal lobes of human subjects diagnosed with early-stage AD. CRMP2 dephosphorylation was observed in human subjects with early-stage AD compared to normal subjects. In this study, the implications of CRMP2 dephosphorylation with respect to AD and potential use as a biomarker and therapeutic intervention in early-stage AD are presented.

The polyglutamine (polyQ) diseases, including Huntington's disease and spinocerebellar ataxias (SCAs), are a group of inherited neurodegenerative diseases which are caused by an abnormal expansion of the glutamine-encoding CAG repeat in each causative gene. The expansion of the polyQ stretch triggers abnormal β-sheet conformational transition and aggregation of polyQ-containing proteins, leading to neurodegeneration. Toward developing therapy for polyQ diseases, we performed a high-throughput screening of a large small chemical compound library for polyQ aggregation inhibitors using an in vitro turbidimetric aggregation assay. As a results, we identified PolyQ Aggregation Inhibitor 1 (QAI1), which is a clinically-approved drug and is known to cross the blood-brain barrier. We revealed QAI1 exerts its anti-aggregation property by inhibiting the upstream toxic β-sheet conformational transition of polyQ proteins. QAI1 was shown to suppress polyQ aggregation and neurodegeneration in Drosophila models of polyQ diseases. Most importantly, we successfully demontrated that oral administration of QAI1 ameliorates the motor dysfunction in two polyQ disease model mice. Furthermore, QAI1 exerted its therapeutic effects even when administered after the symptom onset. We are currently planning a clinical trial to investigate the efficacy of QAI1 as a disease-modifying therapeutic candidate for polyQ disease patients.

Duchenne muscular dystrophy (DMD) is the most common inherited muscular dystrophy. Patients experience DMD in their 20s from cardiac or respiratory failure related to progressive muscle wasting. Currently, the only treatments for the symptoms of DMD are available.Muscle fibrosis, a DMD feature, leads to reduced muscle function and muscle mass, and hampers pharmaceutical therapeutic efficacy. Although antifibrotic agents may be useful, none is currently approved. Phosphodiesterase 4 (PDE4) inhibitors have exhibited antifibrotic effects in human and animal models. In this study,we showed beneficial effects of the PDE4 inhibitor piclamilast in the DMD mdx mouse. Piclamilast reduced them RNA level of profibrotic genes, including collagen1A1, in the gastrocnemius and diaphragm, in the mdx mouse, and significantly reduced the Sirius red staining area. The PDE5 inhibitors sildenafil and tadalafil ameliorated functional muscle ischemia in boys with DMD, and sildenafil reversed cardiac dysfunction in the mdx mouse. Single-treatment piclamilast or sildenafil showed similar antifibrotic effects on the gastrocnemius; combination therapy showed a potent antifibrotic effect, and piclamilast and combination therapy increased peroxisome proliferator-activated receptor-gamma coactivator-1a mRNA in mouse gastrocnemius. In summary, we confirmed that piclamilast has significant antifibrotic effects in mdx mouse muscle and is a potential treatment for muscle fibrosis in DMD.

[Background] Since kidney fibrosis is a common pathway to many kidney diseases, prevention of kidney fibrosis could be a therapeutic strategy for kidney diseases. Previously, we reported that old astrocyte specifically induced substance (OASIS), a transcription factor, promotes proliferation of renal myofibroblasts, analyzed by in vitro assay. However, the pathophysiological significance of OASIS in kidney fibrosis in vivo remains to be elucidated.

[Methods/Results] C57BL/6J mice were subjected to unilateral ureteral obstruction (UUO) to cause kidney fibrosis, analyzed by Sirius red staining and hydroxyproline assay. AEBSF, an inhibitor of OASIS, suppressed kidney fibrosis at day 7 after UUO. Moreover, kidney fibrosis was ameliorated in OASIS KO mice compared with WT mice, accompanied by decreased proliferation of myofibroblasts. To explore the downstream of OASIS, DNA microarray was performed using myofibroblasts from OASIS KO mice. Interestingly, it was found that bone marrow stromal antigen 2 (BST2) was a candidate downstream gene of OASIS. Anti-BST2 antibody treatment attenuated UUO-induced kidney fibrosis.

[Conclusion] OASIS contributes to the develop of kidney fibrosis by promoting the proliferation of myofibroblasts, in part, through increased BST2 expression. OASIS could be a therapeutic target against kidney fibrosis.

Fibroblast-to-myofibroblast differentiation is recognized as critical process of developing irreversible pulmonary fibrosis through the excessive accumulation of extracellular matrix. To elucidate the factor that can induce dedifferentiation of myofibroblast phenotype will provide a new therapeutic target for pulmonary fibrosis. We have previously reported Lung mixed culture-derived epithelial cells (LMDECs) as the cell populations, collected from crude culture of the mouse whole lung, satisfied some characteristics of Type-2 alveolar epithelial cells, and ameliorated experimental pulmonary fibrosis. However, the mechanism as to how LMDECs exerts beneficial effects on the pulmonary fibrosis remains to be clarified. We showed the myofibroblasts dedifferentiation induced by the communication between LMDECs and myofibroblasts. The primary Myofibroblast-Like Cells (MyoLCs), exhibiting increased expression of myofibroblast marker proteins (α-smooth muscle actin and ED-A-fibronectin), were isolated from resected human fibrotic lung. Immunoblotting revealed reduced expression of myofibroblast marker proteins in LMDEC/MyoLC direct contact co-culture, while LMDEC did not change expression of them in non-contact co-culture. We will further investigate the association of LMDEC-derived membrane proteins with myofibroblast dedifferentiation.

Idiopathic pulmonary fibrosis (IPF) is a fatal respiratory disease with an unknown cause and poor prognosis. To precisely know the pathophysiology of lung fibrotic disease, the appropriate animal model correlating to the pathology of IPF is required. Its establishment may contribute to develop a new strategy for the treatment of patients with IPF. From the transcriptome analysis using lung myofibroblasts derived from the patient with IPF, we found the possible correlation between ferroptosis and lung fibrosis. Then, to investigate whether the intracellular overload of ferrous ion in the lung parenchyma induces fibrotic formation, we subpleurally injected ferric chloride solution into the central part of left upper lobe. Within 1 h post-injection, the intracellular elevation of ferrous ion and ROS was detected by berlin blue and dihydrorhodamine, respectively. At 10 days post-injury, diffuse and severe fibrosis occurred in the whole lung lobe, which was well associated with an excessive collagen deposition, an increase in expression of myofibroblast markers and a decrease in static lung compliance. Likewise, the DNA array/gene set enrichment analysis clearly showed the accumulation of extracellular matrix including collagen in the ferric chloride-injured lung. In our model, it is noteworthy that a prominent hyperplasia of type2 alveolar epithelial cells was observed around the fibrotic lesion and that progressive and irreversible fibrogenesis was verified by a follow-up survey at least for 6 weeks. These features correlate to the pathology of IPF but are not observed in other existing mouse models. In conclusion, we established a new reliable lung fibrosis model.

Heart failure is a leading cause of death in developed countries. The role of mRNA regulation in the pathology of heart failure remains elusive. CCR4-NOT complex is a multi-subunit protein complex constituting exonuclease-mediated degradation of poly(A) tails of mRNA, a process called deadenylation. We had previously elucidated that CNOT3, a scaffold subunit of the CCR4-NOT complex, is a crucial regulator of heart function (Cell 2010, Science Signaling 2018). Here we analyzed the roles of deadenylase subunit in heart failure. After 2 weeks of transverse aortic constriction (TAC)-induced pressure overload, expression of CNOT6L deadenylase subunit was markedly upregulated in the hearts. Loss of CNOT6L significantly decreased cardiac contractility and enhanced fibrosis at 2 weeks after TAC. Analyses with transcriptome and CCR4-NOT RIP elucidated that CNOT6L targets mRNA of the GeneX, which stimulates tissue fibrosis. Double knockout of GeneX and CNOT6L improved cardiac fibrosis and dysfunction in single CNOT6 knockout mice. Our ongoing analyses are further elucidating cellular mechanisms for CNOT6L - GeneX axis in heart failure. Thus, CNOT6L deadenylase contributes to prevent progression of heart failure by suppressing expression of fibrotic geneX, implicating a potential therapeutic strategy of targeting mRNA deadenylation.

Pulmonary veins contain a myocardial layer, whose electrical activity is considered to be involved in the genesis of atrial fibrillation. Our previous study revealed that persistent sodium current (late I_Na) contributes to the automaticity of the pulmonary vein myocardium. Class Ⅰ antiarrhythmic drug are used for the treatment of atrial fibrillation, but effects on the automatic activity and the late I_Na of the pulmonary vein myocardium has not been examined. In this study, we investigated the effect of class Ⅰ antiarrhythmics on the automatic activity of the isolated guinea pig pulmonary vein myocardium with microelectrode and voltage clamp experiments. All of the antiarrhythmics examined reduced the maximum rate of rise of tertiapin-induced automatic action potentials. The firing frequency and diastolic depolarization slope were decreased by aprindine, flecainide and propafenone, unaffected by pilsicainide, and increased by cibenzoline and disopyramide. The late I_Na induced by ramp depolarization was reduced by aprindine, flecainide and propafenone, and unaffected by pilsicainide, cibenzoline and disopyramide. These results indicate that class Ⅰ antiarrhythmics have differential effects on automaticity of the pulmonary vein myocardium, and that blockade of the late I_Na results in inhibition of automaticity.

Introduction: Ranolazine has been shown to experimentally and clinically exert an anti-atrial fibrillatory effect, of which electropharmacological profile was not thoroughly assessed to clarify the efficacy.

Methods: Ranolazine dihydrochloride was administered at 0.3 and 3 mg/kg, i.v. to the halothane-anesthetized dogs (n=5).

Results: The low dose increased the heart rate (HR) and cardiac output (CO), whereas no significant change was observed in the mean blood pressure (MBP) or ventricular contraction. It enhanced the atrioventricular conduction, but suppressed the ventricular conduction without any change in the repolarization period. The high dose decreased the HR, MBP, ventricular contraction and CO. It prolonged the repolarization period and T_peak-T_end besides the same effects on the atrioventricular and ventricular conduction as the low dose, but it did not alter the J-T_peakc. It prolonged the atrial (AERP) and ventricular effective refractory period (VERP) by 21 and 29 ms, respectively, giving ΔAERP/ΔVERP of 0.72. Conclusions: Ranolazine has cardiodepressive action along with the ventricular depolarization and repolarization delay. Since ΔAERP/ΔVERP of dronedarone, amiodarone, bepridil and dl-sotalol was reported to be 1.9, 1.6, 1.0 and 1.1, respectively, ranolazine may have a limited efficacy against atrial fibrillation.

Introduction: While a dopamine re-uptake inhibitor vanoxerine suppresses I_Kr, I_Na and I_Ca,L in vitro, its electropharmacological information in vivo is limited.  Methods: Vanoxerine dihydrochloride was intravenously administered at 0.03 and 0.3 mg/kg to the halothane-anesthetized dogs (n=4) under the monitoring of cardiovascular variables.  Results: The low dose increased the heart rate and cardiac output, whereas no significant change was observed in the mean blood pressure, ventricular contraction or pre/afterload.  It prolonged the ventricular effective refractoriness without any change in ECG variables.  The high dose decreased the heart rate, increased the afterload, but it did not alter the other cardiohemodynamic variables.  It delayed the early as well as late repolarization, and equally prolonged the atrial and ventricular effective refractoriness.  No significant change was detected in the intra-atrial, atrioventricular-nodal or intra-ventricular conductions.  Conclusions: Cardio-stimulatory responses after the low dose could be explained by the dopamine re-uptake inhibitory mechanism.  In vivo electropharmacological effects of vanoxerine may largely depend on the I_Kr and I_Na inhibition, whereas I_Ca,L suppression may play a minor role.

Introduction: While lamotrigine is a common antiepileptic drug, it has been reported that overdose of lamotrigine induced the hypotension, cardiac conduction delay, wide complex tachycardia and cardiac arrest. In this study, we tried to bridge the gap between lamotrigine treatment and the onset of these cardiovascular adverse events.

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

Results: The low or middle dose of lamotrigine did not alter any of the variables. The high dose significantly prolonged the PR interval for 45-60 min, QRS width at 10 and 60 min, HV interval at 15 and for 45-60 min, whereas no significant change was detected in the other variables.

Conclusion: Lamotrigine may have a wide safety margin against hemodynamic adverse events since the low dose of 0.1 mg/kg in this study would provide clinically-relevant plasma concentrations. Importantly, the atrioventricular nodal and intraventricular conduction delay indicates toxic dose of lamotrigine may inhibit Ca²⁺ and Na⁺ channels, respectively, which might partly explain clinically-observed cardiovascular adverse events of lamotrigine.

Ischemia/reperfusion (I/R) injury causes ventricular arrhythmia through inducing reactive oxygen species (ROS) and Ca²⁺ overload. We examined the effects of canstatin, a C-terminal fragment of type IV collagen α2 chain, on I/R-induced ventricular arrhythmia. I/R was induced by ligating left anterior descending artery for 10 min. Canstatin (20 µg/kg i.v.) was injected 5 min before the ligation. Ventricular arrhythmia within 10 min after reperfusion was recorded using an electrocardiogram. Neonatal rat cardiomyocytes (NRCMs) or adult rat ventricular myocytes (ARVMs) was subjected to oxygen and glucose deprivation/reoxygenation (OGD/R) in the presence or absence of canstatin (250 ng/ml). ROS production was detected by 2', 7'-dichlorofluorescin diacetate staining. Phosphorylation of Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) was determined by Western blotting. Canstatin significantly decreased duration of I/R-induced ventricular arrythmia without suppressing the incidence. Canstatin significantly inhibited OGD/R-induced ROS production in NRCMs and tended to inhibit OGD/R-induced phosphorylation of CaMKII in ARVMs. This study for the first time demonstrated that canstatin prevents I/R-induced ventricular arrhythmia in part through inhibiting ROS production and CaMKII activation.

Periostin (POSTN), a matricellular protein is related to structural remodeling of pathological heart. However, it remains to be clarified whether POSTN mediates electrical disorders of heart. Thus, we investigated the effects of POSTN on electrophysiological properties in rat ventricular myocytes. Male Wistar rats were injected with recombinant rat POSTN (64 mg/kg, i.v.) for 24 h. After electrocardiogram was recorded, ventricular myocytes were isolated. Action potential (AP) and voltage-dependent Na⁺ channel (Nav) current (I_Na) in the isolated ventricular myocytes or neonatal rat ventricular myocytes (NRVMs) were measured by a whole cell patch-clamp technique. The QRS duration was increased in the POSTN-injected rats. The duration and time to peak of AP were prolonged with a decrease in peak amplitude of AP and I_Na in the isolated ventricular myocytes from POSTN-injected rats. POSTN (1 μg/ml, 24 h) suppressed the peak amplitude of I_Na in NRVMs. The present study for the first time demonstrated that POSTN prolongs the time to peak of AP through inhibiting Nav activity in rat ventricular myocytes. It is suggested that POSTN might cause arrhythmia via prolonging AP duration in ventricular myocytes.

Chronic cerebral hypoperfusion (CCH) is manifested in various CNS diseases accompanied by cognitive impairment. We have previously reported that microglial activation induced excessive inflammatory responses, white matter injury, and resultant aggravation of cognitive impairment in a mouse CCH model with bilateral common carotid artery stenosis (BCAS). Prior to the onset of cognitive impairment, we also observed the increase in the number of GFAP-immunopositive astrocytes at day 14 after BCAS operation. Although the increase remained until day 28, the pathophysiological role of astrocytes in CCH remains to be elucidated. Here, we focused on the pathophysiological significance of the increased number of astrocytes and their regulating mechanisms in CCH. To clarify the involvement of reactive astrocytes in CCH, we checked the subtype of reactive astrocytes, a pro-inflammatory A1-like phenotype or an anti-inflammatory A2-like phenotype, and observed the increase in the expression of A2-like gene in BCAS-operated mice at day 14, whereas no significant difference was seen in the expression of A1-like gene between BCAS- and sham-operated mice. These results imply that anti-inflammatory astrocytes play important roles in the early stage of development in CCH prior to the white matter injury and cognitive impairment.

[Introduction] It is recently reported that sustained activation of inflammasomes (cytosolic protein complexes) in microglia and neural progenitor cells (NPC) may induce neuroinflammation and impaired neurogenesis in neurodegenerative diseases. While liver X receptor (LXR) activation induces transcription of lipid metabolism related genes through a mechanism called transactivation, the activation suppresses expression of genes, such as interleukin-6 (IL-6) and IL-1beta, a mechanism called transrepression. We have developed transrepression-selective LXR ligands which have anti-inflammatory actions without causing hypertriglyceridemia. We determined the effects of transrepression-selective LXR ligands on inflamasome activation of microglia and NPC.

[Materials and Methods] Activation of inflammasomes in 6-3 microglia cell clone or NPC stimulated by TNF and LPS was examined by the expression of inflammasome components (NLRP3 or caspase-1), IL-1beta, or IL-18 using Western blot analysis. The differentiation potential of NPC into neural cells was evaluated by NeuN expression using Western blot analysis. A transrepression-selective LXR ligand (AA70 or M2-76) were pretreated prior to the stimulation by TNF and LPS.

[Results] Stimulation with TNF and LPS induced caspase-1 activation and production of IL-1beta and IL-18 in microglia or NPC. Pretreatment with either AA70 or M2-76 significantly inhibited the inflammasome activation. In addition, stimulation with TNF and LPS significantly suppressed NeuN expression in the differentiated NPC. Pretreatment with either AA70 or M2-76 also significantly inhibited the suppressive effect.

[Conclusion] Pretreatment of microglia or NPC with transrepression-selective LXR ligands inhibited inflammasome activation and suppression of neural differentiation by TNF and LPS.

Since the identification of endogenous H₂S in the mammalian brain in 1989, studies of this molecule uncovered physiological roles in processes such as neuromodulation, vascular tone regulation, cytoprotection against oxidative stress. We previously demonstrated that H₂S induces Ca²⁺ influx in astrocytes by activating transient receptor potential (TRP) channels. During this study we found that H₂S_n activates TRP channels much more potently than does H₂S and that 3-mercaptopyruvate sulfurtransferase produces H₂S₃ and H₂S₂ that activate TRP ankyrin 1 channels Recently, we demonstrated that the chemical interaction of H₂S with nitric oxide (NO) generates H₂S₂ and H₂S₃, and that it gives a mechanism of a synergistic effect between H₂S and NO. Cysteine persulfide (Cys-SSH) together with its glutathione (GSH) counterpart (GSSH) have been proposed to be involved in redox homeostasis. We will also show that 3-mercaptopyruvate sulfurtransferase (3MST) produces Cys-SSH, GSSH, and persulfurated cysteine residues of proteins under physiological conditions together with H₂S_n and H₂S.

We have demonstrated that microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for PGE₂ synthesis, is a critical factor of stroke-reperfusion injury. In this study, we investigated the role of mPGES-1 in neuroinflammation and neurological dysfunctions observed after intracerebral hemorrhage (ICH). Collagenase was injected into the left striatum of adult mPGES-1 knockout (KO) and wild-type (WT) mice. In WT mice, mRNA and protein of mPGES-1 were significantly up-regulated in striatum and cerebral cortex after ICH. In mPGES-1 KO mice, although the hemorrhage and edema size were almost the same as WT mice, survival rate was significantly higher than WT mice. The PGE₂ production, TNF-α induction and glial activation after ICH in mPGES-1 KO brain were significantly less than those in WT brain. DAPI and TUNEL staining showed ICH-induced nuclear condensation and DNA fragmentation in mPGES-1 KO striatum were less than those in WT striatum. Furthermore, mPGES-1 KO mice showed better performance in stepping error test, rotarod test and neurological dysfunction scoring compared with the WT mice. These results suggest that mPGES-1 contributes to ICH-induced neuroinflammation, neuronal apoptosis, neurological dysfunctions and mortality through PGE₂ production. Thus, mPGES-1 may be a new therapeutic target for ICH.

Parkinson's disease is one of the neurodegenerative disorders, caused by progressive degeneration of dopamine (DA) neurons in substantia nigra. Microglial activation by IFNγ/LPS treatment triggers selective loss of DA neurons in midbrain slice cultures. Exosomes are regarded as a novel factor that mediates cell-to-cell interactions. In the present study, we investigated the involvement of exosomes in DA neurodegeneration triggered by microglial activation in rat midbrain slice culture. IFNγ/LPS treatment prominently elevated exosome release from midbrain slice cultures. GW4869, a neutral sphingomyelinase 2 inhibitor, decreased exosome release and prevented IFNγ/LPS-triggered DA degeneration without the inhibition of microglial activation. To directly elucidate the involvement of activated microglial-derived exosome in DA neurodegeneration, we isolated exosomes from culture media of IFNγ/LPS-treated slices and treated them to other slice cultures. Although exosomes from control slices did not affect the survival of DA neurons, exosomes from IFNγ/LPS-treated slices significantly decreased DA neurons. Microglial activation was not triggered by exosomes from IFNγ/LPS-treated slices. These findings suggest that exosomes from activated microglia directly react to neurons and mediate DA neurodegeneration.

The sympathetic nervous system plays critical roles in the differentiation, maturation, and recruitment of immune cells under homeostatic conditions, and in responses to environmental stimuli, although its role in the migratory control of immune cells remains unclear. In this study, using an advanced intravital bone imaging system, we demonstrated that the sympathetic nervous system locally regulates neutrophil egress from bone marrow for mobilization to inflammatory foci. We found that sympathetic neurons were located close to blood vessels in the bone marrow cavity; moreover, upon lipopolysaccharide (LPS) administration, local sympathectomy decreased the velocity of neutrophils, and increased the proportion of neutrophils that remained in place. We also showed that vascular endothelial cells produced C-X-C motif chemokine ligand 1 (CXCL1), which is responsible for neutrophil egress out of bone marrow. Its expression was upregulated, and was suppressed by β-adrenergic receptor blockade, resulting in inhibition of neutrophil egress into the systemic circulation. Furthermore, systemic β-adrenergic signaling blockade decreased the recruitment of neutrophils in the lung under acute systemic inflammation. Taken together, the results of this study demonstrated a new regulatory system, wherein local sympathetic nervous activation promoted neutrophil egress by inhibiting Cxcl1 expression in bone marrow endothelial cells in a β-adrenergic signaling-dependent manner, contributing to the recruitment of neutrophils at the onset of inflammation.

Brown adipose tissue (BAT) is a primary site for non-shivering thermogenesis in mammals. In cold temperature, β-adrenergic stimulation activates mitochondrial uncoupling protein 1 (UCP1), which consequently generates heat by uncouples of the respiratory chain. Previous report suggested that intracellular Ca²⁺ signaling induced by transient receptor potential vanilloid 2 (TRPV2) activates UCP1 in BAT thermogenesis. However, molecular mechanisms of intracellular Ca²⁺ signaling in BAT are not well characterized. We have been systematically studying the physiological functions of Na⁺/Ca²⁺ exchangers, which regulate intracellular Ca²⁺ signaling. Recently, we found that Na⁺/Ca²⁺ exchanger type 1 (NCX1) is abundantly expressed in interscapular BAT (iBAT) from wild-type mice. Therefore, in this study, our research interest focuses on elucidating functional involvement of NCX1 in BAT thermogenesis. Intriguingly, we observed that NCX1-heterozygous (NCX^+/-) mice did not maintain their core body temperature in cold exposure. Furthermore, UCP1 transcripts in iBAT were significantly decreased in NCX^+/- mice compared with wild-type mice. These results suggest that NCX1 may contribute to BAT thermogenesis against cold environment.

The intermediate-conductance Ca²⁺-activated K⁺ channel K_Ca3.1 is involved in the promotion of tumor growth and metastasis, and is a potential therapeutic target for cancer. Higher K_Ca3.1 gene expression correlates with the shorter overall survival in cancer patients. Histone deacetylases (HDACs) post-translationally regulate the expression and activity of a number of proteins that play a crucial role in cancer development and progeression. We have showed that the K_Ca3.1 expression and activity are potentially reduced by the treatment with class I HDACs, HDAC2 and HDAC3 in prostate and breast cancer cell lines. Hypoxic tumor microenvironment is a common characteristic of solid cancers, and is associated with cancer metastasis and poor cancer prognosis. Hypoxia up-regulates the class III HDAC, SIRT1. Here we investigated the effect of the SIRT1 inhibitor on the K_Ca3.1 expression and activity in several types of cancer cells using real-time PCR, western blotting, flow cytometry, and voltage-sensitive fluorescence dye imaging assays. Pharmacological and siRNA-mediated inhibition of SIRT1 down-regulated K_Ca3.1 transcription and reduced its activity. These results suggest that SIRT1 may be a potential therapeutic target for K_Ca3.1-overexpressing cancers.

Androgen deprivation (castration) therapy andwith anti-androgens have become standardremain the clinical mainstay of treatments for metastatic and aggressive prostate cancer (PCa). Yet, these therapies are limited by the inevitable onset ofmost PCa patients will progress to castration-resistant PCa (CRPC) for several reasons such as the overexpression of androgen receptors (AR). Thus, it is necessary to develop Novel therapies are desired for the advanced PCa. Ca²⁺-activated K⁺ channels (K_Ca) are key molecules inregulate cancer cell behaviors including proliferation progression. We revealed the predominant expression of K_Ca2.2 in human androgen-sensitive prostate cancer cell line, LNCaP cells, using a real-time PCR, western blotting, and whole-cell patch clamp recording. The treatment with UCL1684, a K_Ca2.x channel blocker inhibited the store-operated Ca²⁺ entry in LNCaP cells, resulting in suppressive effect on proliferation of LNCaP cells. The pharmacological or siRNA-mediated inhibition of AR for 48 hr decreased the expression level levels of K_Ca2.2 transcripts in LNCaP cells, whereas the UCL1684-induced inhibition of K_Ca2.2 activity did not affect the expression levels of AR transcripts, suggesting that K_Ca2.2 is a downstream effector of AR signaling in LNCaP cells. The short-term androgen deprivation for 48 hr decreased the K_Ca2.2 protein expression, whereas the long-term one for 96 hr increased it. Together, K_Ca2.2 might be a possible therapeutic candidate in castration-resistant PCa.

LAT1 overexpressed in many cancer cells is an attractive therapeutic target because it provides cancer cells with essential amino acids required for proliferation. JPH203, a specific competitive inhibitor for LAT1, has been reported to suppress proliferation of cancer cells. Recently, we found that anti-tumor effects of JPH203 between MDA-MB-231 cells, a model of highly malignant breast cancer cells (=TNBC) and T-47D cells(=non-TNBC) were different. To investigate the difference in JPH203 sensitivity, we analyzed global gene expression change in JPH203-responsive T-47D cells in the presence or absence of JPH203. Among them, we focused on ATF4, a master transcription factor for stress response, which activates anti-apoptotic pathway. Knockdown of ATF4 in MDA-MB-231 cells, enhanced JPH203 induced cell death. Furthermore, some reports showed that ATF4 elicite CTH, the biosynthetic enzyme for cysteine, which protects TNBC from nutrient stress. Knockdown of CTH in MDA-MB-231 cells, also enhanced growth inhibition by JPH203 treatment. These results suggest that nutrient stress caused by JPH203-treatment activated anti-apoptotic signaling via ATF4 in MDA-MB-231 cells and that CTH inhibition could be a novel way of breaking resistance to anti-LAT1 therapy in MDA-MB-231 cells.

Aquaporin-5 (AQP5) is a water-selective channel protein expressed in alveolar epithelial and submucosal gland cells, and plays an important role to maintain water homeostasis in the lung. Recently, several lines of evidence indicated that AQPs regulate not only plasma membrane water permeability, but also various cellular functions, such as cell migration and growth. In our previous study, we have found that AQP5 potentiated TNF-α-induced cytokine expression, whereas it attenuated Th2 cytokine-induced response. In the present study, we first examined the underling mechanisms involved in the attenuation by AQP5 in Th2 cytokine signaling. In AQP5-expressing cells, the IL-13-induced phosphorylation of STAT6 was lower than that in control cells. Consistent with this, phosphorylation of JAK1 and TYK2 by IL-13 in AQP5 expressing cells were considerably lower than those in control cells, suggesting that AQP5 inhibits IL-13 and tyrosine kinase complex. In addition, we have established transgenic mouse in which AQP5 is highly expressed in the lung, to investigate the pathophysiological role of modification of cytokine signaling by AQP5. The phenotypes of this transgenic mice will be also shown this presentation.

Magnesium ion (Mg²⁺) is an essential divalent cation and cellular Mg²⁺ concentration is tightly regulated by various Mg²⁺ channels/transporters. Therefore, dysfunction of Mg²⁺ channels/transporters may lead to a variety of cardiovascular or neuromuscular disorders. TRPM7 is a non-selective cation channel, which predominantly permeates Mg²⁺ under physiological conditions. We generated tissue-specific transgenic mouse models expressing the dominant negative TRPM7 mutant (TRPM7DN-Tg) to study the physiological and pathophysiological mechanisms of Mg²⁺ regulation. Whole-cell patch-clamp recordings revealed that TRPM6/7 currents in HEK293 cells were almost completely attenuated by co-expression of TRPM7DN mutant. Renal tubule-specific TRPM7DN-Tg exhibited dysregulation of serum Mg²⁺ level and urinary Mg²⁺ excretion. Interestingly, in these mice, phenylephrine (PE)-induced vascular contractile responses was significantly attenuated. On the other hand, vascular smooth muscle-specific TRPM7DN-Tg showed attenuation of PE-induced contractile responses without changing serum Mg²⁺ level and urinary Mg²⁺ excretion. These results suggest that TRPM6/7 channels are tissue-dependently involved in the regulation of Mg²⁺ homeostasis and vascular contraction. Our tissue-specific TRPM7DN-Tg will be useful animal models for studying magnesium disorders.

Atherosclerosis often results in high incidence of vascular occlusion and has been recognized as the major cause of coronary artery disease. We had previously reported that promoter polymorphism of myocardin-related transcription factor A (MRTF-A) is associated with coronary atherosclerosis. However, the contribution of MRTF-A to the development of atherosclerosis remains unclear. Macrophages are known to be important mediators of atherosclerosis. In this study, we found that MRTF-A was highly expressed in lesional macrophages in human carotid atherosclerotic plaque. To investigate the role of macrophagic MRTF-A in the pathogenesis of atherosclerosis, we generated ApoE null MRTF-A transgenic mice (ApoE^−/−/MRTF-A^tg/+), in which human MRTF-A was specifically overexpressed in monocytes/macrophages. We found that ApoE^−/−/MRTF-A^tg/+ aggravated atherosclerosis and accumulated prominent lesional macrophages in the aortic sinus. We also found that MRTF-A promoted proliferation of macrophages and mitigated apoptosis both in vitro and in vivo due to downregulation of the expression of cyclin-dependent kinase inhibitors. Taken together, our data indicated that MRTF-A contributes to the development of atherosclerosis by modulating functional properties of pro-atherogenic macrophages.

Prostaglandin E₂ (PGE₂) is pro-inflammatory and immunomodulatory lipid mediator formed from PGH2 by microsomal Prostaglandin E Synthase-1 (mPGES-1). PGE₂ binds EP receptors, EP1-EP4, and induces pharmacological function. We analyzed what type of EP receptor is most important for recovery from ischemia.

Method) Male 6-8 weeks old C57Bl/6N (wild type=WT), EP4WT and EP4 receptor deficient mice (EP4KO) were used. Ischemic hind limb model was made by femoral artery ligation. Blood flow recovery was estimated by leaser Doppler images. Angiogenesis was estimated by expression of CD31, TGF-beta and SDF-1 by using immunohistichemical analysis and real time PCR. Contribution of regulatory T cells (Tregs) was estimated by immunohistochemical study and real time PCR against FOXP3 expression, that was specific transcript factor for Tregs. 

Results) Expression of EP4 receptor in the ischemic muscle was enhanced compared to other EP receptors. Selective EP4 antagonist significantly suppressed recovery from ischemia compared to vehicle treated mice. Furthermore, blood flow recovery was significantly suppressed in EP4KO compared to EP4WT. Sixty seven percentage of EP4KO showed necrosis in ligated foot in contrast, no necrosis lesion was seen in EP4WT. The number of accumulated FOXP3⁺ cells in the ischemic muscle was decreased in EP4KO compared to WT. Expression of TGF-beta and SDF-1 were suppressed in EP4KO. Moreover, EP4KO transplanted with WT-Tregs (CD4⁺CD25⁺) significantly enhanced blood flow recovery compared to EP4KO transplanted with non-Tregs (CD4^-CD25⁺).

Those results suggested that PGE₂-EP4 signaling induced recovery from ischemia via accumulating Tregs. Highly selective EP4 agonist might be useful for treating peripheral artery disease.

In smooth muscle cells (SMCs), caveolin (cav)-1, an essential component of caveolae, forms Ca²⁺ microdomain accumulating voltage-dependent Ca²⁺ channels (VDCC) and ryanodine receptors (RyR). The functional coupling between VDCC and RyR causes SMC contraction, i.e. excitation-contraction (E-C) coupling. On the other hand, Ca²⁺ influx through VDCC activates Ca²⁺/calmodulin-dependent protein kinase (CaMK), and promotes gene transcription in neurons, i.e. excitation-transcription (E-T) coupling. E-T coupling is known in SMCs, but its structural basis and physiological function are unknown. Therefore, we examined the relationships between Ca²⁺ microdomain formed by caveolae and E-T coupling in SMCs. When the mesenteric artery was depolarized, the phosphorylation of CREB in the nuclei of SMCs and induction of c-fos was detected. These responses were not observed in the tissue of Cav-1 KO mouse that lacks caveolae in SMCs and those in which caveolae were destroyed by methyl b cyclodextrin. The CREB phosphorylation was significantly attenuated by a CaMKK2 inhibitor STO609 and CaMK2 inhibitor KN93. Furthermore, fluorescence imaging analyses detected a direct molecular coupling between cav1 and CaMKK2. These results suggest that caveolae accumulate Ca²⁺ channels and CaMKK2 and cause not only E-C coupling but also E-T coupling in SMCs.

The mechanism of serotonin (5-HT)-induced vasoconstriction and intracellular Ca²⁺ ([Ca²⁺]i) mobilization is not completely elucidated. 5-HT-induced vasoconstriction partly involves Ca²⁺-independent activation of Rho kinase. However, the mechanism of Rho kinase activation by 5-HT is still unknown. We examined the mechanism of 5-HT-induced [Ca²⁺]i mobilization of rat aortic smooth muscle cells using microscopic fluorometry. We also investigated whether 5-HT-induced constriction in rat thoracic aortas is mediated by Rho kinase activation through Src, epidermal growth factor receptor (EGFR), and extracellular signal-regulated kinase (Erk).

5-HT induced a biphasic [Ca²⁺]i response, and the initial [Ca²⁺]i increase was attenuated by inositol triphosphate (IP₃) receptor blocker, and inhibitors of Src and phosphoinositide 3-kinase (PI3K), but not L-type Ca²⁺ channel blocker (LCBB). The second [Ca²⁺]i increase was attenuated by LCBB. Contractile response to 5-HT significantly attenuated by inhibitors of Rho kinase, Erk1/2, Src, and EGFR. These data suggest that 5-HT induces Ca²⁺ release from the endoplasmic reticulum via Src and PI3K, and subsequently extracellular Ca²⁺ influx via L-type Ca²⁺ channel, and 5-HT-induced constriction is mediated by Rho kinase activation via Src, EGFR, and Erk in rat thoracic aortas.

Guanine-rich DNA and RNA can form a four-stranded structure, termed G-quadruplexes (G4) in cells. The formation of G4 is implicated in many physiological events, such as gene transcription, translation, and epigenetics. However, the presence of G4 has not been revealed in the brain. Here, we demonstrate the localization of G4 in the mouse brain by immunohistochemical analysis. In cultured mouse forebrain neurons, numerous punctate G4 immunoreactivities (G4-IR) were observed in nuclei as well as in cytoplasmic areas, including axons, dendrites, and postsynapses. Interestingly, the G4-IR in nuclei show more co-localizations with the bright spots of DAPI-positive heterochromatin clusters in cultured mature compared to immature neurons. In slices from adult mouse brain, the G4-IR were distributed throughout the brain but were particularly prominent in the hippocampus, olfactory bulb, and cerebellum. In the hippocampus, G4-IR were strongly expressed in neurons and weak in astrocytes. Consistent with the results in cultured neurons, the nuclear G4-IR were co-localized with heterochromatin in calbindin-positive mature granule cells but less in doublecortin-positive neuronal progenitor cells in the dentate gyrus. Electron microscopic immunolabeling revealed G4-IR on nucleolus-associated chromosomal domains (NADs) and cytoplasm in the adult mouse hippocampal CA1 region. These observations demonstrate the critical roles of G4 in neuronal developmental stages through chromatin conformational changes and in the cytoplasmic metabolism of RNA.

G-quadruplexes (G4) are noncanonical four-stranded nucleic acid structures formed by guanine-rich sequences. Recently, a number of studies have demonstrated that RNAs containing G4 structures are involved in biological and pathological processes, including transcription, mRNA maturation, translation, and their relevance to G4-binding proteins. However, the detail function of G4 RNAs in neuron still elusive. In this study, to identify G4 RNAs in brain, we performed an RNA immunoprecipitation sequencing (RIP-seq) using a G4-specific antibody, named G4 RIP-seq. We found several mRNAs of genes that are likely relevant to neuronal function or diseases. In fact, those mRNA has several G-rich sequence motifs and we successfully determined 3D G4 structures of those motifs in vitro. Immunochemical assays of certain G4-binding protein, together with in situ hybridization of mRNAs suggested that cellular G4 RNA might be relevant to neuronal or neuro-physical function. In this presentation, we will discuss the recent results of this study.

Autism spectrum disorder (ASD) is one of neurodevelopment disorders, with impairment of social behaviors as a major hallmark. Cumulating body of evidence has implicated the neuroinflammatory system as a contributing factor in the pathology of ASD. The objective of this study was to investigate the expression of prostaglandin EP3 receptor mRNA in the brain of mice ASD model. The litters born to valproic acid-treated mothers were tested for their social interaction at the age of 5-6 weeks old. Upon completion of behavioral observation, the mice were sacrificed and assessed for the expression of brain EP3 receptor mRNA. Behavioral results showed shorter duration of sniffing behavior in mice born to VPA-treated mothers. Further examination in this group of mice revealed significantly lower expression of EP3 receptor mRNA in the area prefrontal cortex and hippocampus. The present study suggests that the molecular brain mechanism involved in the arachidonic acid cascade is essential to the pathophysiology of ASD.

Schizophrenia, a severe psychiatric disorder, exhibits three major symptoms, including cognitive dysfunction. Piccolo, a presynaptic protein, plays a role in synaptic vesicle trafficking, and is suggested to be associated with several psychiatric disorders in the postmortem and GWAS analyses. We previously proposed that mice knocking down Piccolo in the prefrontal cortex (PFC-Piccolo-KD mice) were useful as an animal model for schizophrenia based on those face and predictive validities. In this study, we investigated the neuronal circuits that contribute to cognitive dysfunction in the new animal model.

We firstly confirmed the bidirectional connection between the PFC and ventral hippocampus (vHIP) via the perirhinal cortex (PRC) by using neuronal tracer. Cognitive dysfunction in the PFC-Piccolo-KD mice in the novel object and location recognition test was recovered by activation of the PFC-PRC circuit. Furthermore, mice knocking down Piccolo in the PRC showed impairment of cognitive memory, and its impairment was also ameliorated by activation of the PRC-vHIP circuit.

These findings suggest that disturbance of the PFC-PRC-vHIP neuronal network is involved in schizophrenia-like symptoms. Accordingly, improvement of disturbed PFC-PRC-vHIP network could be a therapeutic approach for cognitive dysfunction in schizophrenia.

Recent studies reported that human psychiatric disorders display oligodendroglial abnormalities and alterations in oligodendrocyte structure. Our previous studies suggested that 5-HT_1A receptor in the hippocampus may be involved in the protection of myelin loss induced by unadaptable excessive stress. In oligodendrocytes, adenomatous polyposis coli (APC) appears at maturation and the onset of myelination. In the present study, we investigated whether 5-HT_1A receptor regulate remyelination in unadaptable stress-induced myelin damage. A single exposure to restraint stress for 60 min induced a decrease in head-dipping behavior in the hole-board test. This stress response disappeared in mice that had been exposed to repeated restraint stress for 60 min/day for 14 days. In contrast, repeated exposure to restraint stress for 240 min/day for 14 days did not develop stress adaptation, and still showed a decrease in head-dipping behaviors. Immunohistochemistry analysis revealed that the expression level of APC was decreased in the dentate gyrus of the hippocampus of stress-maladaptive mice. These behavioral and biochemical changes were inhibited by chronic treatment with flesinoxan, a 5-HT_1A receptor agonist. The present findings indicate that activation of 5-HT_1A receptor may promote remyelination in stress-induced myelin damage.

Temporal lobe epilepsy with distributed hippocampal seizure foci is often intractable and secondary generalization of its seizures might lead to sudden death. Early termination of the seizures through spatially extensive hippocampal intervention is not feasible directly, due to its large size and irregular shape. In contrast, the medial septum (MS) is a promising target to govern hippocampal oscillations through its divergent connections to both hippocampi. Combining this ‘proxy intervention' concept and precisely-timed stimulation, we report here that closed-loop MS electrical stimulation can quickly terminate intrahippocampal seizures and suppress their secondary generalization in a rat kindling model. Precise stimulus timing governed by internal seizure rhythms in a closed-loop manner was essential for the seizure terminating effect. Cell-type-specific optogenetic stimulation revealed that alternating activation of MS GABAergic and glutamatergic neurons within the internal seizure rhythms can underlie the seizure terminating effect. This seizure rhythm congruent MS electrical stimulation can be directly translated into clinical application.

Parkinson's disease is a neurodegenerative disorder cased by loss of nigrostriatal dopaminergic neurons. For over 40 years, levodopa had been established as a gold standard for PD treatment. However, long-term treatment with levodopa is often complicated by the development of adverse effects such as abnormal involuntary movements (AIMs), referred to as levodopa-induced dyskinesia (LIDs). We here report the pharmacological effects of the intrastriatal infusion of memantine, a non-competitive N-methyl-D-aspartate (NMDA) receptor antagonist, in a 6-OHDA-lesioned mouse model of hemiparkinsonism. Spontaneous and apomorphine-induced rotational activities with an abnormal hind limb stepping were assessed as Parkinsonian symptoms. Daily intraperitoneal injection of levodopa (15 mg/kg) was performed for 21 days, with assessing AIMs score as an index of LID development. Intrastriatal memantine infusion targeted into the right dorsal striatum, using an iPRECIO^TM programmable micro infusion pump with a brain infusion kit, was examined with 4 doses (3 days/dose). Infusion of memantine significantly alleviated Parkinsonian symptoms, and it also reduced AIMs score with a dose-dependent manner. These results support the idea that over-activation of the striatal NMDA receptor function might generate both Parkinsonian symptoms and LIDs.

【Introduction】It has been reported that hyperbilirubinemia increase the risk of psychiatric disorder including schizophrenia (Miyaoka 2000) though the molecular mechanisms are not yet well understood. Several reports show the Gunn rat, which is an animal model of congenital hyperbilirubinemia, has agitative like behaviors (i,e,. Hayashida 2009). Recently, our research group have reported that risperidone improve agitative like behaviors in Gunn rat. The risperidone strongly antagonized serotonin receptor (5HT2AR). In this study, we investigated serotonin neurotransmission in the Gunn rat.【Methods】It was investigated whether 5HTAR specific antagonist (Ketanserin) injection improve behavioral abnormality in Gunn rats. The amounts of serotonin and its metabolites in the Gunn rat were measured by high performance liquid chromatograph, furthermore, serotonergic neurons in the dorsal raphe nucleus were visualized by immunohistochemistry.【Results】Ketanserin injection improved the hyperactivity and agitation like behaviors in Gunn rats. There were significantly higher serotonin and its metabolite at the frontal cortex in the Gunn rats compared to the control rats. The immunohistochemistry showed that the number of TPH positive cells was increasing in dorsal raphe nucleus of Gunn rats.【Conclusion】The serotonergic dysfunctions in the cortical regions seem to play an important role in hyeprbilirubinaemia associated abnormal behaviors. Our study suggests that intervention with abnormal serotonergic transmission may improve symptom of hyeprbilirubinaemia associated psychosis.

Objective: Diagnostic criteria for rare diseases are often revised with increased numbers of cases, because the pathogenesis of such diseases is complicated and heterogeneous. Therefore, nationwide surveillance is needed to clarify the pathogenesis of rare diseases. In this study, we applied AI (Artificial Intelligence) to diagnose neurodegenerative disorders, such as multiple system atrophy (MSA) and spinocerebellar degeneration (SCD).

Methods: We constructed an AI diagnostic system based on Chainer. After machine learning, diagnostic probability (0–1.0) was estimated for each case. Medical records of cases involving patients with MSA and SCD were provided from the Ministry of Health, Labour and Welfare. 4,949 cases involving patients with MSA and 7,073 cases involving patients with SCD between 2004 and 2008 were used for this study.

Results: Diagnostic probabilities of SND (Striata-Negra Degeneration) and OPCA (Olivo-Ponto-Cerebellar Atrophy) were estimated at 0.97 and 0.88. In contrast, the probability of SDS (Shay-Drager Syndrome) was lower than that of SND and OPCA. Diagnostic probabilities of sSCD, AD_SCD, or SP (Spastic paraplegia) were estimated at 0.95, 0.86, and 0.83. On the other hand, the probabilities of AR_SCD and Other_SCD were estimated at 0.04 and 0.03. 

Conclusion: An AI diagnostic system could correctly categorize cases with SND, OPCA, sSCD, AD_SCD, and SP. Although cases involving patients with familial SCD required genetic testing, those with AD_SCD were correctly estimated by the AI diagnostic system.

To achieve more effective cancer treatment, we have established and analyzed "clinically relevant radioresistant (CRR) cells" that can survive exposing to 2 Gy/day X-rays for more than 30 days. CRR cells show resistance against hydrogen peroxide (H₂O₂) that is one of the reactive oxygen species. However, the resistant mechanism to H₂O₂ has not been elucidated yet. Therefore, we investigated the involvement of iron ion in the resistant mechanism to H₂O₂ in CRR cells because iron ion has been reported to react with H₂O₂ and produce hydroxyl radical (･OH). ･OH have been shown to react with plasma membrane phospholipid and lead to cell death. Internal Fe²⁺ and ･OH amount were decreased in CRR cells compared with its parental cells. In addition, expression of ferritin, which is iron-binding protein, was increased in CRR cells. No internal H₂O₂ increase and no lipid peroxidation were seen in CRR cells after 50 µM H₂O₂ treatment for 2 hours, whereas internal H₂O₂ uptake and lipid peroxidation was increased after 50 µM H₂O₂ treatment for 2 hours in parental cells. Furthermore, Pretreatment of 10 µM of FeCl₂ leads to more cell death after administration of 50 µM H₂O₂ in CRR cells. Administration of phospholipid also led to further cell death after 50 µM H₂O₂ treatment in CRR cells. These results suggest that intracellular Fe²⁺ content is very important against oxidative stress response in CRR cells and control of Fe²⁺ amount may be an effective option for cancer that is resistant to treatment.

Baicalin and baicalein are the flavonoids derived from the dried roots of Scutellaria baicalensis Georgi, known as baikal skullcap roots (Huang Qin) in the traditional Chinese medicine. Several biological activities of these two compounds have already been reported such as anti-inflammatory, anti-allergic, and choleretic activities. In this study, we investigated an anti-leukemic activity of baicalin and baicalein against a human acute myelocytic leukemia cell line, HL-60 cells. HL-60 cells were incubated with baicalin and baicalein for several days. Cell number was counted after the incubation. Both baicalin and baicalein decreased the number of cells in a dose-dependent manner. Baicalein, a deglycosylated form of baicalin showed intense suppressive effects at lower concentrations than baicalin. Since both baicalin and baicalein showed anti-leukemic activities within a short time, we assessed the killing activities of these two compounds using a CCK-8 kit. Both baicalin and baicalein showed killing activities at higher concentrations within 24 h. Observation under a microscope of the cells incubated with these two compounds for 3 h revealed deformation of the cells i.e. abnormal cell shapes such as swelling and blebbing, suggesting that the cells exposed to these compounds went into necrosic cell death. We still continue to investigate the precise mode of action.