Facilitation of fear extinction is expected to shorten the duration of treatment for fear-related disorders. Previously, we found that selective agonist of δ-opioid receptor (DOP), KNT-127, facilitates extinction learning of contextual fear. Here, we investigated the brain regions which mediate the action of KNT-127 on fear extinction in mice. On day 1, male C57BL/6J mice were contextually conditioned with 8 foot-shocks. On day 2, the mice were re-exposed to the conditioning chamber for 6 min as an extinction training (re-exposure 1). KNT-127 was microinjected into the amygdala (AMY), hippocampus (HPC), prelimbic (PL) and infralimbic (IL) sub-regions of the medial prefrontal cortex, 30 min before re-exposure 1. On day 3, mice were re-exposed to the chamber for 6 min as a memory testing (re-exposure 2). As a result, KNT-127 (50 ng/mouse) into the AMY and IL, but not HPC and PL, significantly reduced freezing behavior in re-exposures 1 and 2. These effects of KNT-127 in the AMY and IL were abolished by pretreatment with a selective DOP antagonist naltrindole (NTI). Further, MEK/ERK inhibitor, U-0126, blocked the effect of KNT-127 in the AMY. These results suggested that KNT-127 facilitated extinction learning via DOPs in the AMY and IL, and that MEK/ERK pathway in the AMY mediates the extinction-facilitating action of KNT-127.

We propose that L-DOPA by itself is a neurotransmitter. Recently, a G-protein-related receptor (GPCR) GPR143, a gene product of ocular-albinism1, was identified as an L-DOPA receptor. We previously showed that non-effective dose of L-DOPA potentiates behavioral response to quinpirole, a dopamine D2 receptor (D2R). However, it remains undetermined whether and how GPR143 regulates D2R-mediated behaviors. In this study, we analyzed behavioral responses to several D2R ligands using Gpr143 gene-deficient (GPR143-KO) mice. We found that haloperidol, a D2R antagonist (0.5mg/kg)-induced catalepsy was attenuated in GPR143-KO mice when compared to wild type (WT) mice. To clarify which neuron circuits are responsible for this phenotype, we investigated haloperidol-induced catalepsy using mice that expressing cre recombinase in D2R-, adenosine A2a receptor (indirect pathway)-, choline acetyltransferase (cholinergic interneuron)-positive neurons. Haloperidol-induced catalepsy was attenuated in D2R-cre (+); Gpr143^flox/y and ChAT-cre; Gpr143^flox/y mice. These results suggest that GPR143 expressed in the striatal cholinergic interneurons plays an important role in haloperidol-induced catalepsy.

Hachimijiogan (HJG) is traditional herbal medicine. Recently, it has been reported that cognitive dysfunction in patients with Alzheimer's disease (AD) was improved by HJG treatment. However, the mechanism by which HJG improves cognitive dysfunction is still unclear. Senescence-accelerated mouse prone 8 (SAMP8) mice are used as a spontaneous animal model of AD. The present study examined the effect of HJG on cognitive dysfunction and glial cell marker expression in SAMP8 mice.

SAMP8 mice were orally administered HJG (1000 mg/kg/d) from 12 weeks of age. Three-chamber sociability and social novelty test were conducted at 38-39 weeks of age. The glial cell marker levels in the hippocampus were analyzed by western blotting.

Vehicle-treated SAMP8 mice showed the impairment of social cognition compared with SAMR1 mice, which are resistant to senescence. On the other hand, HJG-treated SAMP8 mice did not show significant impairment compared with SAMR1 mice. The levels of arginase-1, a protective microglia marker, were significantly decreased in vehicle-treated but not HJG-treated SAMP8 mice.

Our findings suggest that social cognitive dysfunction of SAMP8 mice is due, in part, to the reduced protective microglia. HJG may make the progression of cognitive dysfunction slower by attenuating reduction in protective microglia.

Chemotherapy-induced peripheral neuropathy (CIPN) is a complication caused by several anti-cancer drugs, which profoundly affects the patient's quality of life. Paclitaxel (PTX) is used in the treatment of common cancers, and usually causes dysesthesias, paresthesias and numbness, hypersensitivity to mechanical stimuli, that patients frequently suffer in feet and hands. Mirogabalin (MGB) has been developed as a novel gabapentinoid, and its analgesic effect is exerted by binding to the α₂δ-1 subunit of voltage-gated calcium channels. Although MGB is used for the treatment of peripheral neuropathic pain including diabetic peripheral neuropathy and postherpetic neuralgia, no clinical studies have been reported in CIPN. Here, we conducted an investigate the effects of MGB on PTX-induced peripheral neuropathic pain. A single oral administration of MGB dose-dependently inhibited PTX-induced mechanical allodynia but did not affect locomotor activity. Next, we administered MGB topically and found that intrathecal injection suppressed mechanical allodynia, but intradermal injection into footpad did not. In fact, α₂δ-1 protein expression was increased in the spinal cord on the PTX model. Together, these results suggest MGB inhibits PTX-induced mechanical allodynia by acting on α₂δ-1 subunit in the spinal dorsal horn.

Dry eye-induced chronic pain involves hypersensitivity and hyperalgesia, and is a clinically serious problem. However, effective therapeutic approach has not been established other than eye drops to alleviate the symptoms. Aiming at developing a pathogenic mechanism-based therapeutic strategy, we performed experiments using a rat dry eye model with lacrimal gland excision (LGE). On the LGE side, corneal hypersensitivity and hyperalgesia were developed. In the trigeminal nucleus of the LGE side, neuronal hyper-activation, transient activation of microglia, persistent activation of astrocytes, upregulation of the voltage-dependent Ca²⁺ channel α₂δ-1 subunit were observed. Next, we evaluated the efficacy of ophthalmic treatment for corneal damage and pregabalin, a ligand for α₂δ-1 subunit, after chronic pain was established in LGE rats. Ophthalmic treatment alone was not effective for hyperalgesia. In contrast, the combination of ophthalmic treatment and pregabalin effectively abrogated hyperalgesia, neural activity, the upregulated α₂δ-1 subunit, and activated astrocytes. These results highlight a crucial role of α₂δ-1 subunit upregulation in the trigeminal nucleus as a pathogenic mechanism and the therapeutic target for dry eye–induced chronic pain.

Chronic itch is an unpleasant sensation and reduces quality of life. Especially, atopic dermatitis (AD) is representative disease with chronic itch. The itching associated with a nettle rash is potently alleviated by H₁ receptor antagonists, but that with AD is not. Nevertheless, its underlying mechanisms are poorly understood. Here, using AD model mice, we showed inhibitory effect of the novel gabapentinoid, mirogabalin on spontaneous scratching behavior. The number of scratch bouts were increased in AD model mice as compared with healthy mice. These spontaneous scratching were not suppressed by H₁ receptor antagonist. Next, we examined the effect of mirogabalin (10 mg/kg) by oral administration in AD model mice, and mirogabalin suppressed scratch bouts. We also examined sedation by using healthy mice with Rota-Rod test and showed mirogabalin (10 mg/kg) did not have sedation. Furthermore, we examined the effect of other gabapentinoids. Gabapentin (100 mg/kg) and pregabalin (30 mg/kg) also inhibited scratch bouts in AD model mice.  These results suggest that mirogabalin may be effective against chronic pruritus in AD.

Mechanical allodynia is one of the symptoms of neuropathic pain and is produced by tactile stimulation. Recently, we have identified a subset of spinal dorsa horn (SDH) inhibitory interneurons that is operated by adeno-associated viral (AAV) vectors incorporating a neuropeptide Y promoter (AAV-NpyP⁺). In a model of neuropathic pain caused by peripheral nerve injury (PNI), these neurons exhibit deeper resting membrane potentials, and their excitability is impaired, which are necessary for neuropathic allodynia. However, the mechanism underlying these changes remains unknown. In this study, we show that the dysfunction of AAV-NpyP⁺ neurons require astrocytes that are activated in the SDH after PNI. We found that inhibition of PNI-induced activation of SDH astrocytes by expressing a dominant negative form of STAT3 (dnSTAT3: an inactive mutant STAT3) alleviated Aβ fiber-derived neuropathic allodynia and normalized alterations in resting membrane potentials and excitability of AAV-NpyP⁺ neurons. Our findings suggest that suppressing activation of astrocytes after PNI restores normal AAV-NpyP neurons activity and attenuates neuropathic allodynia. Thus, inhibiting astrocytes activation may be a new therapeutic target for neuropathic allodynia.

It is well known that acute exposure to physical stress produces a transient antinociceptive effect (called stress-induced analgesia [SIA]). One proposed mechanism for SIA involves noradrenaline (NA) in the central nervous system. NA has been reported to activate inhibitory neurons in the spinal dorsal horn (SDH), but its in vivo role in SIA remains unknown. In this study, we found that an antinociceptive effect on noxious heat after acute exposure to restraint stress was impaired in mice with a conditional knockout of α_1A-adrenaline receptors (α_1A-ARs) in inhibitory neurons (Vgat-Cre;Adra1a^flox/flox mice). A similar reduction was also observed in mice treated with DSP-4, a selective neurotoxin for NAergic neurons in the locus coeruleus (LC). Furthermore, whole-cell patch-clamp recordings using spinal cord slices revealed that NA-induced increase in the frequency of spontaneous inhibitory postsynaptic currents in the substantia gelatinosa neurons was suppressed by silodosin, an α_1A-AR antagonist, and by conditional knockout of α_1A-ARs in inhibitory neurons. Moreover, under unstressed conditions, the antinociceptive effects of intrathecal NA and phenylephrine on noxious heat were lost in Vgat-Cre;Adra1a^flox/flox mice. Our findings suggest that activation of α_1A-ARs in SDH inhibitory neurons, presumably via LC-NAergic neurons, is necessary for SIA to noxious heat.

Synucleinopathies are neurodegenerative diseases caused by aggregation of α-Synuclein (α-Syn). While it has been suggested that pathogenic α-Syn can spread in the whole brain like a prion protein, those molecular mechanisms are still unknown. Here, we show that RNA G-quadruplexes (G4RNAs) have an important role in α-Syn aggregation. We found that α-Syn binds to guanine-enriched RNA sequences using RNA Bind-n-seq in vitro. In addition, α-Syn preferentially formed a complex with G4RNAs than with other RNA secondary structures. Under the molecular crowding conditions, α-Syn underwent liquid-liquid phase separation (LLPS), and G4RNAs significantly facilitated liquid-to-solid transition of α-Syn. In α-Syn overexpressing cells, α-Syn preformed fibril (PFF) increased G4RNA foci and in turn formed α-Syn aggregates. Furthermore, α-Syn aggregates were colocalized with G4RNA foci in the dopaminergic neurons of α-Syn PFF-injected mice. These observations suggest that G4RNA is a key factor of α-Syn aggregation and cell-to-cell transmission under the pathological condition. We are trying to reveal the mechanisms underlying increases of G4RNA foci by cellular stress, and define endogenous G4RNA forming RNAs involved in α-Syn phase transition.

Expanded CAG/CTG triplet repeats are causal in a number of human neurological disorders, and can be classified into two types according to the location of the repeats; 1) The CAG repeat expansion in coding regions, including Huntington's disease (HD), spinocerebellar ataxia (SCA) type-1, 2, 3, 6, 7, and 17, spinal and bulbar muscular atrophy (SBMA), and dentatorubral pallidoluysian atrophy (DRPLA). 2) The CAG/CTG repeat expansion in noncoding, especially in 3' untranslated regions (3'-UTRs), including myotonic dystrophy type 1 (DM1) and SCA8. Here, we show a DNA targeting compound, cyclic Pyrrole-Imidazole Polyamide (cPIP) can suppress the pathogenesis of coding and noncoding CAG/CTG repeat expansion diseases. cPIP bound to duplex as well as hairpin CAG/CTG DNA specifically, inhibiting the RNA polymerase II passage in a repeat length dependent manner in vitro. cPIP inhibits the CAG/CTG repeat-derived mRNA transcript, result in reduction of pathogenic CUG-RNA foci and polyglutamine (polyQ) accumulations. This study presents a candidate compound for targeting pathogenic expanded CAG/CTG repeat DNA, demonstrating the concept of lowering levels of repeat disease-causing RNAs and proteins.

The amyloid-beta peptide (Aβ), a major component of senile plaques, is believed to be the underlying trigger of the development of Alzheimer's disease (AD). The elucidation of mechanism(s) that induce Aβ overproduction from its type I transmembrane precursor protein (APP) is therefore important for the development of effective AD therapies. Semaphorin3A (Sema3A), a secreted type of repulsive axon guidance molecule, is implicated in the development of various neurodegenerative diseases. It was reported that Sema3A and its signaling molecules accumulated and aggregated in the brain of AD patients. However, the molecular link between Sema3A signaling and AD pathogenesis remains unknown. Here, we provide evidence regarding the interaction between APP and PlexinA, a Sema3A receptor component, through their extracellular regions. We also narrowed down the interacting regions to 100 amino acid or less. Based on these findings, we are now investigating whether the APP-PlexinA interaction affects APP function and metabolism, which might provide novel insights into involvement of Sema3A signaling in overproduction of Aβ.

Aims: We have already shown that the alteration of polyunsaturated fatty acid (PUFA) in the hypothalamus is involved in the regulation of pain. However, how become the changes in composition of hypothalamic PUFA under pain state remain unclear. It is reported that three fatty acid-binding protein (FABP) subtypes, which regulate PUFA intracellular trafficking and signal transduction, are expressed in the mammalian brain. In this study, we confirmed the changes in the expression of FABP3 in the median eminence, which is part of the hypothalamus, of postoperative pain model mice.

Methods: Paw incision-induced postoperative methods were adopted as a pain model in male ddY mice. Mechanical hypersensitivity was examined by the von Frey test. The mRNA analysis of FABP subtypes were measured by real-time PCR, and cellular localization of its protein level were measured by immunofluorescent study.

Results: Postoperative pain mice elicited mechanical allodynia on day 2 after paw incision, and mRNA expression of FABP3 significantly increased in the hypothalamus of the postoperative pain model mice compared to that in control mice. FABP3 positive cells in the median eminence colocalized with Iba-1 positive cells, which is a microglial cell marker, but not neuron and astrocyte marker. Its protein level significantly increased in the median eminence on day 2 after paw incision and returned to the control level on day 4 after paw incision. 

Conclusions: Our results suggest that FABP3 in the median eminence may change in pain stimuli and may be a key molecule to control pain signaling.

Prevention of oxaliplatin-induced peripheral neuropathy (OIPN) by thrombomodulin alfa (TMα) involves thrombin-dependent activation of thrombin-activatable fibrinolysis inhibitor (TAFI) and protein C (PC), in addition to inactivation of high mobility group box 1 (HMGB1). We thus analyzed the anti-OIPN effects of activated forms of TAFI (TAFIa), known as carboxypeptidase B (CPB), and PC (APC) in mice. OIPN was inhibited by TMα, an anti-HMGB1-neutralizing antibody (HAb) or TAFIa/CPB, and partially by APC. Combination of HAb with APC, but not TAFIa/CPB, at subeffective doses abolished OIPN. TAFIa/CPB at a subeffective dose in combination with APC at a maximal dose also abolished OIPN. Intraplantar (i.pl.) HMGB1-induced allodynia was inhibited by TMα, but not APC or TAFIa/CPB. TAFIa/CPB abolished the allodynia following i.pl. C5a, a complement component, and bradykinin, known to be degraded by TAFIa/CPB. The C5a-induced allodynia was also inhibited by HAb as well as a C5aR antagonist. The C5aR antagonist, but not combination of B1 and B2 antagonists, abolished OIPN. Our study ascertains that thrombin-dependent degradation of HMGB1 and generation of  APC and TAFIa/CPB by TMα are necessary to abolish OIPN, and provides novel evidence that C5a targeted by TAFIa/CPB contributes to the development of OIPN via HMGB1-dependent mechanisms.

Hsp90 is a molecular chaperone that contributes to the activation and stabilization of client proteins. In our previous studies, we found that an inhibition of Hsp90 reduced cardiac remodeling during the development of heart failure in rodents. Simvastatin, an antihyperlipidemic drug, was shown to inhibit Hsp90. However, it is still unclear whether simvastatin attenuates cardiac remodeling via inhibition of Hsp90. Therefore, we investigated effects of simvastatin on the development of heart failure following myocardial infarction in rat. The results showed that treatment of the animals with simvastatin attenuated the development of cardiac fibrosis. Furthermore, we found that simvastatin attenuated the interaction of Hsp90 with c-Raf and calcineurin and decreased their contents. These results suggest that Hsp90 inhibition by simvastatin treatment is, at least in part, responsible for the reduction in the development of myocardial remodeling following acute myocardial infarction.

Heart failure (HF) is a principal cause of death and disability in industrialized countries. Since cardiomyocyte hypertrophy and myofibroblast differentiation are caused during the progression of HF, the suppression of these processes is considered to be therapeutic strategy. The aim of this study is to determine the effect of a PRMT5 selective inhibitor EPZ015666 (EPZ) on left ventricular dysfunction.

Primary cultured cardiomyocytes from neonatal rats were treated with EPZ and stimulated with phenylephrine (PE). PE-induced cell hypertrophy was significantly suppressed by the treatment with EPZ. During cardiomyocyte hypertrophy, various fetal gene expression is induced. PE-induced increase in the expression of hypertrophic genes was significantly inhibited by EPZ treatment. EPZ also suppressed transforming growth factor-beta (TGF-b)-induced myofibroblast differentiation in cultured cardiac fibroblasts. Next to examine the effect of EPZ on pressure overload-induced heart failure in vivo, we used the transverse aortic constriction (TAC) surgery in the mouse. Echocardiographic analysis showed that TAC-induced left ventricular hypertrophy and dysfunction were significantly improved by treatment with EPZ.

These data indicate that the pharmacological inhibition of PRMT5 suppresses pressure overload-induced pathological HF.

Heart failure with preserved ejection fraction (HFpEF) is a common syndrome in the elderly population, especially in women. To elucidate the mechanism of HFpEF, we investigated the effects of aging and sex on cardiac function in a "two-hit" HFpEF model, which combines metabolic and mechanical stress (Nature. 568, 351-356, 2019). We induced HFpEF in 6-month-old male- and 2-year-old male- and female mice by "two-hit" model and examined the effects of aging and sex on cardiac function. Cardiac function was evaluated by echocardiography at the beginning 0, 5, and 15 weeks of the experiment. Before the treatment, aged mice showed mild cardiac hypertrophy and reduced left ventricular contractility compared to the young mice, and there was no clear sex difference in cardiac function in aged mice. At 5 weeks, young mice showed diastolic dysfunction and cardiac hypertrophy. In sharp contrast to young mice, some aged mice showed markedly reduced left ventricular contractility. Additionally, some aged mice showed symptoms of heart failure at 15 weeks, showing more varied consequences than the young mice. In particular, some mice developed heart failure with reduced ejection fraction, which was not observed in young mice. Collectively, our results demonstrate the age-related differences in the response to the "two-hit" HFpEF model.

Background: We previously reported that natural compound curcumin suppresses cardiomyocyte hypertrophy and the development of heart failure via inhibiting p300 histone acetyltransferase (HAT) activity. In this study, we investigated the effect of curcumin analogue, GO-Y022 on p300-HAT activity, cultured cardiomyocyte hypertrophy and heart failure in vivo.

Methods & Results: In vitro HAT assay using recombinant p300-HAT domain showed that GO-Y022 inhibited p300-HAT activity as well as curcumin. Primary cultured cardiomyocytes prepared from neonatal rats were treated with curcumin or GO-Y022 and stimulated with phenylephrine (PE). 1 µM GO-Y022 suppressed the following results to the same extent as 10 µM of curcumin: PE-induced histone acetylation, hypertrophic response gene transcription, and cardiomyocyte hypertrophy. Finally, 8-week-old C57BL/6J male mice were subjected to transverse aortic constriction (TAC) surgery and orally administrated GO-Y022 or curcumin for 8 weeks. Cardiac echography indicated that a low dose of GO-Y022 (1 mg/kg) repressed TAC-induced increase in left ventricular posterior wall dimension and decrease in Fractional shortening to the same extent as 50 mg/kg of curcumin.

Conclusion: GO-Y022 may be used for heart failure therapy at a lower dose than curcumin.

BACKGROUND: Several studies have reported that patients treated with sunitinib, a tyrosine kinase inhibitor, have developed left ventricular dysfunction and heart failure, but there is currently no treatment for heart failure with sunitinib. The purpose of the present study is to identify candidate drugs for the treatment of sunitinib-induced heart failure using a large medical database. METHOD: We analyzed the FDA Adverse Event Reporting System (FAERS) and the WHO global adverse event reporting database (VigiBase) to find candidate drugs for prevention of sunitinib-induced heart failure. The effects of the candidate drugs on cell viability and cell morphology were evaluated using the WST-8 assay and immunostaining in H9c2 cells derived from rat cardiac rhabdomeres. RESULTS: FAERS and VigiBase searches revealed significantly higher reporting odds ratio (ROR) of heart failure in patients treated with sunitinib than in those not treated with sunitinib. The ROR was reduced by concomitant use of Vitamin D (FAERS: ROR 0.50, 95% CI 0.26-0.96; VigiBase: ROR 0.37, 95% CI 0.10-0.95). In vitro, Vitamin D significantly improved the viability and maintained the cell morphology in H9c2 cells exposed to sunitinib. CONCLUSION: The findings suggest the potential value of Vitamin D in preventing sunitinib-induced heart failure.

Carvedilol, a nonselective β-adrenergic receptor blocker, has been reported to improve endothelial dysfunction in patients with cardiovascular diseases and in a diabetic animal model but that mechanism of action is unknown. The purpose of this study was to investigate the effect of carvedilol on the endothelial-response of aortas from diabetic mice and the underlying mechanism. Vascular reactions and protein expressions were measured in aortas isolated from both control and nicotinamide and streptozotocin-induced diabetic mice (DM). UK14304 (UK)-induced endothelial-dependent relaxation declined along with the decrease of nitric oxide (NO) levels in aortas from DM. Carvedilol prevented the inhibition of UK-induced relaxation and NO production caused by DM. The phosphorylation of Akt and endothelial nitric oxide synthase (eNOS) were very low in UK-stimulated DM aortas compared with those of control group. Treatment with carvedilol significantly increased not only the phosphorylation of Akt and eNOS under UK-stimulation but also the AMP-activated protein kinase (AMPK) phosphorylation in the DM. In conclusion, carvedilol significantly ameliorated the endothelial dysfunction in DM aortas, in which increased NO levels through Akt/eNOS activation, up-regulated AMPK phosphorylation may be involved.

Aortic dissection is a severe aortic disease, in which the aortic wall is separated into two layers at the medial level, resulting in two lumens being created, a true and a false lumen. Most cases have a sudden onset resulting in death. Therefore, it is required to establish a preventive strategy. Eucommia ulmoides leaf (EUL) extract contains various flavonoids such as quercetin, chlorogenic acid, geniposidic acid, and so on, and it is suggested to have a protective effect against cardiovascular diseases. In this study, we investigated the preventive effect of EUL on the onset of aortic dissection.

We generated pharmacologically-induced aortic dissection model mice (LAB model). In C57Bl / 6J mice, three agents are administered; (1) nitric oxide synthase inhibitor (L-NAME) that causes vascular endothelial damage, (2) angiotensin II (Ang II) that causes hypertention, and (3) lysyl oxidase inhibitor (BAPN) that causes medial fragility. EUL extract was orally administered daily throughout the experiment.

Hypertension, caused by Ang II+BAPN loading was significantly suppressed by EUL. In the LAB model, macrophage infiltration into the aortic wall was increased, but it was suppressed by EUL administration. As a result, EUL showed the preventive effects against the onset of aortic aneurysm, dissection, and death from rupture.

[Background and Purpose] We have reported that myocardial ischemia was observed by anaphylaxis (The 94th Annual Meeting of the Pharmacological Society). In this study, we examined the effect of Suramin, an antagonist of both P2X and P2Y2 receptors, on electrocardiogram ST-segment elevation and hemodynamics during anaphylaxis induction.

[Method] Rats received Suramin 100 or 300 micro-M or physiological saline as a control 30 minutes prior to the induction of anaphylaxis following administration of compound 48/80 (C48/80). Blood pressure and electrocardiography recording were started before Suramin administration and continued until 30 minutes after anaphylaxis induction and assessment.

[Results] ST-segment elevation was observed after C48/80 administration, which was significantly suppressed by each dose of Suramin pretreatment. After induction of anaphylaxis, heart rate, systolic and diastolic blood pressure, and rate pressure product (RPP) decreased which was significantly suppressed by pretreatment with 300 micro-M of Suramin.

[Consideration] These results suggest that pretreatment with Suramin may partially improve the hemodynamic deterioration due to anaphylaxis.

【Introduction】 Evaluation of drug-induced cardiotoxicity is important to avoid adverse effects, such as arrhythmia and contractile dysfunction, which is considered to result in heart failure, in non-clinical and clinical studies. For example, BMS-986094, which was developed as a Hepatitis C virus nucleotide polymerase (non-structural 5B) inhibitor, was withdrawn from phase 2 clinical trials because of unexpected heart failure by long-term administration. Although animal models have been widely used to assess cardiac contraction, in vitro models are expected to assess human-specific contraction. We have previously developed the methods to assess cardiac contraction using motion analyses in human iPS cell-derived cardiomyocytes (hiPSC-CMs). Here we assessed whether BMS-986094 induced chronic contractile dysfunction in hiPSC-CMs.

【Methods】 We used iCell cardiomyocyte 2.0 (CDI). Motion analyses were performed using a cell motion imaging system (SI8000, Sony). Calcium imaging was performed using Fluo-8/AM.

【Results】 We found that BMS-986094 decreased both contraction and relaxation velocity by 96 h exposure, while acute exposure with BMS-986094 had little effects. In contrast, sofosbuvir, which has the same drug target, had little effect during 6-day exposure. Next, we analyzed the effect on calcium transient. BMS-986094 decreased calcium transient at 96 h, while sofosbuvir did not affect.

【Conclusion】 Thus, the imaging analysis of hiPSC-CMs would be useful to assess the chronic contractile dysfunction in human.

Tanshinone VI (TanVI) is prepared from Salvia miltiorrhiza Bunge, which has been widely used for treatment of cardiovascular disease in Chinese medicine. In our previous study, we showed the differentiation effect of TanVI on cardiosphere-derived cell (CDC) prepared from adult rat cardiac tissue. However, the exact mechanism of TanVI underlying the differentiation from cardiac stem cells to cardiomyocytes is yet unclear. In this study, we examined the effect of TanVI on the intracellular signaling pathway in CDCs during myocardial differentiation. After CDCs in the presence of TanVI were cultured, the cells expressed a cardiomyocyte marker cardiac troponin T. Concomitantly, an increase in the expression of the cardiac transcription factor Nkx2.5 was also observed in TanVI-treated CDCs. Treatment of the cells with TanVI resulted in reduction of GSK3β phosphorylation. The β-catenin expression level in CDCs was also decreased. Furthermore, TanVI reduced the phosphorylation levels of Erk1/2. These results suggest that TanVI enhances the differentiation of CDCs into cardiomyocytes via an attenuation of GSK3β,β-catenin and Erk1/2 phosphorylation.

Hereditary renal hypouricemia type-1 (RHUC1) is a rare disease associated with markedly lower plasma urate (UA) and increased fractional excretion of UA (FE_UA) due to URAT1 dysfunction, and exercise-induced acute kidney injury (EIAKI) is known to be a serious complication, but its pathogenesis is unknown. The aim of this study is the investigation of pathogenesis of EIAKI and the effect of topiroxostat, a non-purine-type xanthine oxidoreductase inhibitor (XOI) using high HPRT activity Urat1-Uox double knockout (DKO) mice establishing as novel animal model of RHUC1. DKO mice were used in a forced swimming test as loading exercise to explore the onset mechanism of EIAKI and evaluate related purine metabolism and renal injury parameters. In DKO mice, exercise exacerbated renal injury and functional markers, and increased urinary UA/Cr ratio and plasma UA. In addition, NLPR3 inflammasome activation and increased IL-1β were observed in the kidney. Finally, we demonstrated that topiroxostat improved renal injury and functional parameters of EIAKI. The pathogenic mechanism of EIAKI was found to be due to increased levels of IL-1β via NLRP3 inflammasome signaling associated with excessive urinary UA excretion. In addition, topiroxostat, a non-purine-type XOI, appears to be a promising therapeutic agent for the treatment of EIAKI.

Histamine receptor H₃ (H₃R), a G_i/o-coupled receptor, is dominantly expressed in the central nervous system and regulates neurotransmitter release. Our previous study showed that H₃R was also expressed in the pancreatic β cells and had an inhibitory effect on glucose-induced insulin secretion from MIN6 cells, a cell line from mouse pancreatic β cells. However, the in vivo roles of H₃R on β cells in the regulation of plasma insulin level are still unknown. In the present study, we generated and phenotyped β cell specific H₃R knockout mice (cKO mice) to elucidate the importance of H₃R for glucose homeostasis. Blood glucose testing showed that H₃R deletion from β cells resulted in the lower glucose levels after glucose challenge due to higher insulin secretion. Glucose-induced insulin secretion from isolated cKO islets was higher than that from control islets. These data demonstrated that disruption of H₃R led to higher insulin secretion in vivo. Immunohistochemical analysis showed that the number of Ki67-positive β cells was augmented in cKO islets. Morphometric analysis revealed the increased number and size of islets in cKO pancreas. These data indicated that proliferation of β cells was enhanced by H₃R deletion. In conclusion, H₃R in pancreatic β cells has negative impact on insulin secretion and their proliferation.

Aging causes cognitive and motivational declines, but the biological basis remains elusive. Here we analyzed distinct behavioral effects of aging in C57BL6N (B6N) and C57BL/6J (B6J) strains. In this study, mice first learned a visual discrimination task to obtain food rewards by responding to the correct one of two visual stimuli. Then, they learned a response direction task of responding to either left or right for food rewards. Attentional set-shifting, behavioral flexibility between the tasks, is known to depend on working memory. Aged B6N mice showed motivational declines in both tasks. By contrast, task motivation was intact in aged B6J mice, but some of them showed a deficit in attentional set-shifting. We also analyzed synaptic proteomes in the medial prefrontal cortex, a brain region crucial for attentional set-shifting. Young and aged B6J mice showed differential expression of many synaptic proteins, some of which increased only in a subset of the aged mice with attentional set-shifting intact. These findings suggest that different biological mechanisms related to genetic and synaptic factors underlie motivation and cognitive declines with aging.

[Background] Autophagy in the skeletal muscle maintains muscle mass. The NAD⁺-dependent deacetylase SIRT1 positively regulates autophagy. Here, we examined whether SIRT1 in the skeletal muscle maintains muscle mass via promoting autophagy.

[Methods and Results] We obtained tibialis anterior muscle (TA) from 60-weeks old wild-type mice (WT) and muscle-specific SIRT1 knockout mice (SIRT1MKO). Western blotting showed that acetylated lysine levels were increased in SIRT1MKO compared with WT, suggesting suppressed SIRT1 activity in SIRT1MKO. Histological analysis by HE staining showed that the myofiber diameter was 9% smaller in MKO than WT. The percentage of central nuclei, an indicator of muscle regeneration, was 19% higher in SIRT1MKO than WT. To test autophagic activity in the muscle, we treated mice with colchicine, an inhibitor of autophagosome degradation, and measured markers of autophagosomes including LC3-II/LC3-I ratio (Western blotting) and the level of LC3 dots (Immunostaining). At baseline, LC3-II/LC3-I ratio and LC3 dot levels were unchanged in MKO. However, colchicine treatment increased the LC3-II/LC3-I ratio and the LC3 dot level in WT but not in SIRT1MKO, suggesting suppression of autophagic flux in SIRT1MKO. Levels ubiquitinated proteins, which are degraded by autophagy, was also increased in SIRT1MKO.

[Conclusion] These results suggest that SIRT1 plays a critical role in maintenance of skeletal muscle mass via positive regulation of autophagy.

Introduction: Misfolded tau aggregates associated with clinical syndromes and various neurodegenerative diseases. To date, many second-generation of tau PET tracers were developed to overcome the limitation of the first-generation such as off-target binding. Recently, we developed [¹⁸F]SNFT-1 from compound optimization of [¹⁸F]THK-5351, which showed high affinity for monoamine oxidase-B (MAO-B) as off-target binding. The aim of study was to compare the binding properties of [¹⁸F]SNFT-1 and second-generation tau PET radiotracers to human brain tissues.

Methods: In vitro competitive binding assay were performed for tau aggregates, amyloid aggregates, and recombinant MAO-A and MAO-B. After preparing ¹⁸F-labeled compounds, in vitro autoradiography was performed using frozen human brain sections with immunostaining of phosphorylated tau (tau IHC) for evaluation of binding selectivity.

Results and Discussion: Although second-generation of tau PET radiotracers showed a similar binding affinity for tau aggregates, the off-target binding affinity was different. SNFT-1 showed a high binding affinity for tau aggregates, which was comparable to MK-6240, with no interaction of amyloid aggregates as well as MAO enzymes. In vitro autoradiography demonstrated that distribution of radiotracer's binding was similar to tau IHC in the medial temporal area of Alzheimer's disease. On the other hands, second-generation tau PET radiotracers showed little binding to the frontal cortex in a case of progressive supranuclear palsy that contains high density tau aggregates.

Conclusion: [¹⁸F]SNFT-1 would be a promising candidates for imaging tau aggregates. Further binding characterization is required to validate the binding of non-AD tau aggregates.

[Aim] Lower urinary tract symptoms due to intrapelvic nerves damage are frequent complications of pelvic surgery. Previously, we reported that bilateral injury to accessory nerves (ACN), which extend from the major pelvic ganglion, resulted in bladder fibrosis and bladder dysfunction in rats within 72 h until at least 4 weeks post-surgery. Prevention of bladder fibrosis is necessary to maintain bladder function. Herein, we examined the mechanisms underlying bladder fibrosis associated with ACN injury.

[Methods] Ten-week-old male Wistar/ST rats were categorized into sham and bilateral ACN injury (BAI) groups. In the BAI group, the ACN was crushed for 1 min using reverse-action tweezers on both sides. After 4, 8, 12, 24, and 72 h, we examined bladder histology using Masson's trichrome staining and evaluated mRNA expression levels of TGF-β1 and MCP-1 using real-time PCR analysis.

[Results] Increased collagen area-to-total bladder area ratio of BAI groups was noted at 12, 24, and 72 h postoperatively, compared to sham groups. Postoperative mRNA expression levels of TGF-β1 and MCP-1 at 12, 24, and 72 h, and 4, 8, 12, and 24 h respectively, were higher in BAI groups compared to sham groups.

[Conclusion] Upregulation of MCP-1 followed by TGF-β1 may be involved in bladder fibrosis associated with ACN injury.

Hypothesis. Overflow urinary incontinence (OUI) often occurs as a complication after pelvic surgery. Our previous experiment found that bone marrow-derived stem cell lysate (BMSCL) improves neurogenic erectile dysfunction. In this study, we investigated the effectiveness of BMSCL in a model of OUI by bilateral accessory nerve injury (ACNI). 

Methods. Rat bone marrow-derived stem cells were collected, and BMSCL (lysate of 1×10⁶ cells/PBS) was prepared. Eight-week-old male Wistar-ST rats were divided into sham+PBS (sham, n=7), ACNI+PBS (ACNI, n=10), and ACNI+BMSCL (BMSCL, n=10) groups. Following surgery, PBS or BMSCL (100μl/body) was administered intravenously. Bladder function, bladder weight, and morphology were evaluated after one week. In addition, the response of carbachol (CCh) was assessed.

Results. While 7 of 10 rats in the ACNI group showed symptoms of OUI, only 3 of 10 rats in the BMSCL group presented symptoms of OUI. Bladder weight in the ACNI group was significantly larger than in the sham group (P<0.01), while bladder weight in the BMSCL group was significantly lower than in the ACNI group (P<0.01). Fibrotic area in the ACNI group was larger than in the sham group, while that in the BMSCL group was smaller than in the ACNI group. The maximum response to CCh in bladder specimens in the ACNI group was higher than that of sham group, while this was lower in the BMSCL group than in the ACNI group.

Conclusion.  BMSCL improved bladder function and morphology, suggesting that intravenous injection of BMSCL may be a useful treatment for neurogenic bladder dysfunction.

OBJECTIVE: Cisplatin-induced acute kidney injury (AKI) is well known, and the nephrotoxicity of cisplatin restricts its clinical application. Currently, there are no drugs are recommended for the prevention of cisplatin-induced AKI. Forced hydration and diuresis may partially prevent nephrotoxicity of cisplatin, but it is still difficult to entirely prevent kidney injury. Thus, establishment of a new preventive method against cisplatin-induced AKI is required. Therefore, in this study, the purpose of this study was to clarify the efficacy of sodium valproate in cisplatin-induced AKI.

METHODS: In order to establish cisplatin‐induced AKI animal model, C57BL/6 mice were administered with either cisplatin (15 mg/kg, i.p.) or saline (control). The degree of renal damage was assessed by various renal function parameters and pathological evaluation. The effect of sodium valproate on cisplatin-induced cytotoxicity was evaluated using HK2 cells, MKN-1 cells and LLC cells.

RESULTS: Cisplatin treatment worsened various renal function parameters and tubular damage scores, which were significantly improved by co-treatment with sodium valproate. The decrease in cell viability of HK2 cells by cisplatin was significantly improved by co-treatment with sodium valproate. On the other hand, sodium valproate had no adverse effect on the reduction of cell viability of various cancer cells by cisplatin.

CONCLUSIONS: The results of this study indicated that sodium valproate could act as a potential preventive drug for cisplatin-induced AKI.

We have reported protective effect of NaHS [hydrogen sulfide (H₂S) donor] on cyclophosphamide (CYP)-induced rat bladder dysfunction by improving CYP-induced shortening of intercontraction intervals (ICI) and increases in non-voiding contractions (NVCs). In this study, we examined mechanisms of the protective effect. Nine-week-old male Wistar rats were pretreated with NaHS (10 μmol/kg, ip) or saline once daily for 7 days. CYP (150 mg/kg, ip) or saline had been injected 2 days before urodynamic experiments, and after the experiments, bladder tissues were collected to perform HE staining. In some rats, vehicle or capsaicin (CAP, 125 mg/kg, sc), which can desensitize CAP-sensitive afferent nerves, was pretreated 4 days before urodynamic experiments. In bladder tissues, CYP increased neutrophile infiltration, bleeding, and edema, but NaHS partially improved only the edema. CAP prolonged ICI and reduced NVCs in CYP-treated rats. NaHS-induced improvement of CYP-induced ICI shortening and NVC increasing was not detected in CAP-treated rats. These data suggest that NaHS showed protective effect on bladder dysfunction in CYP-treated rats via suppression of CAP-sensitive bladder afferent nerves but not of bladder inflammation.

Cancer cells require more nutrients than normal cells to maintain rapid growth and proliferation. L-type amino acid transporter 1 (LAT1: SLC7A5) is highly upregulated in various cancers, and is regarded as a molecular target for cancer therapy. LAT1 preferentially transports large neutral amino acids (Leu, Ile, Val, Met, Phe, Tyr, Trp, and His) including many essential amino acids in a Na⁺-independent manner. JPH203, an LAT1-specific high-affinity inhibitor, strongly suppresses cancer cell proliferation and tumor growth. However, the contribution of LAT1 to the amino acid uptake in cancer cells and the effect of JPH203 on cellular protein synthesis have not been established. Here, we revealed that JPH203 drastically suppresses the uptake of large neutral amino acids into pancreatic cancer cell lines, regardless of the presence or absence of Na⁺. This indicates that LAT1 has the main contribution to cellular amino acid uptake among amino acid transporters including Na⁺-dependent transporters. Furthermore, we revealed, by polysome profiling analysis, that JPH203 treatment suppresses cellular protein synthesis. These results indicate that LAT1 constitutes the main uptake pathway for large neutral amino acids in cancer cells.  Inhibition of LAT1 efficiently suppresses their uptake and reduces the protein synthesis in cancer cells.

Understanding the molecular mechanism of neuronal differentiation is extremely important to overcome the incurable diseases caused by nervous system damage. Neurite outgrowth is essential for neuronal differentiation and regeneration, and cAMP response element-binding protein (CREB) is one of the key transcriptional factors positively regulating this process. Neuronal differentiation stimuli activate mammalian target of rapamycin complex 2 (mTORC2) / Akt signaling to phosphorylate CREB. However, the molecular mechanism that regulates the activity of this signaling remains poorly understood. We found that neuronal differentiation stimuli increased a protein level of protein phosphatase 6 (PP6), a member of type 2A Ser/Thr protein phosphatases in N2a cells and mouse ES cells. The decrease in autophagy activity was suggested to be involved in the stimulation-induced increase in PP6 expression. PP6 knockdown suppressed mTORC2/Akt/CREB signaling and failed neurite outgrowth. SIN1 is a unique component of mTORC2, and dephosphorylation of SIN1 increases the activity of mTORC2 against Akt. We found PP6 knockdown increased SIN1 phosphorylation. These data suggest that PP6 may positively regulate neurite outgrowth by dephosphorylating SIN1 to activate mTORC2/Akt/CREB signaling.

Sulfides, such as Na₂S and NaHS, enhance Ca_v3.2 T-type Ca²⁺ channel activity, thereby promoting pain signals. We hypothesize that, as does L-Cys, a thiol compound, sulfides might interact with zinc binding to His-191 in Ca_v3.2 and cancel the zinc-induced channel inhibition. On the other hand, the effects of polysulfides, such as Na₂S₃ and Na₂S_4, on Ca_v3.2 function have yet to be investigated. Thus, we compared the effects of sulfides and polysulfides on T-channel-dependent currents (T-currents) in human Ca_v3.2-expressing HEK293 cells, and analyzed possible involvement of sulfide-zinc interaction. Na₂S and NaHS at 3-30 µM rapidly caused remarkable and persistent increase in T-currents. In contrast, Na₂S₃ and Na₂S₄ in the same range caused only slight and transient T-current increase, followed slowly by its decrease below the baseline. In the presence of tricine, a weak Zn²⁺ chelator, T-currents increased, which was reversed by ZnCl₂ at 30 µM. The increased T-currents in the presence of tricine was not altered by Na₂S at 30 µM, but augmented by addition of Na₂S following ZnCl₂. Our data suggest that the sulfide-induced enhancement of Ca_v3.2 function involves the interaction of sulfide with Zn²⁺ possibly binding to His-191 in Ca_v3.2, and cannot be mimicked by polysulfides.

Histamine is a well-known autacoid, which widely exists in vertebrate tissues and plays various roles. Its physiological functions depend on the activation of four types of histamine-related G protein-coupled receptors (H1R, H2R, H3R, H4R). Especially, H1R is expressed ubiquitously, which is involved in most histamine-induced allergic effects.

Previous functional studies reported that H1R mainly activates G_q protein, and the following optogenetical and chemical studies revealed that inhibition of histaminergic neurons induce acute non-REM sleep. However, recent studies suggested that H1R activates four types of G protein (G_s, G_i/o, G_q, and G_12/13), and thus the signaling axis for non-REM sleep remains elusive.

Structure-guided mutgenesis is a desirable tool for extracting specific signaling, which helps the understanding of the histamine signaling mechanism. Here, we determined the histamine-bound H1R-G_i structure at 3.4 Å resolution. This structure allowed clear assignment of histamine-H1R-G_i, which reveals the signaling axis of H1R-G_i. Moreover, our structure revealed that H1R adopts the G_i-specific binding mode, which differs from the G_q-coupled H1R structure reported previously.

Our results provide a molecular mechanism for the signal switch and a molecular basis for the further development of histamine drugs.

Melatonin (N-acetyl-5-methoxytryptamine) activates melatonin receptors (MT₁ and MT₂), which are one of the G_i-coupled class A GPCRs and transduce inhibitory signaling by inhibiting the adenylyl cyclase (AC). Melatonin thus induces our sleep and modulates our circadian rhythm, and melatonin receptors have long been regarded as an important therapeutic target for an insomnia. Although melatonin itself serves as a sleep-inducing supplement, its property is not enough to use clinically, because it is rapidly cleared from our body. Therefore, a lot of melatonin analogs with prolonged release properties have been developed so far, such as ramelteon, agomelatine, tasimelteon. 

Recently reported crystal structures of ligand-bound MT₁ and MT₂ elucidated the structural basis of ligand entry and recognition, but the molecular mechanism of the ligand-induced MT₁ structural change that would lead to G_i-coupling remains unclear.

Here we report the cryo-EM structure of the MT₁-G_i signaling complex at 3.3 Å resolution. The structure reveals the receptor activation mechanism, in which the ligand-induced conformational changes are propagated to the G-protein coupling interface. As compared to other G_i-coupled receptors, MT1 exhibits a large outward movement of TM6, which is considered to be a specific feature of G_s-coupled receptors. The structural comparison among the G_i- and G_s-complexes demonstrated the conformational diversity of the C-terminal entry of the G_i protein, suggesting the loose and variable interactions at the helix end. These notions, together with our biochemical and computational analyses, highlight the different binding modes of Gαi and provide the basis for the selectivity of G-protein signaling.

SET is a multifunctional protein that acts as an intrinsic inhibitor of the tumor suppressor protein phosphatase 2A and a histone chaperone. Increased SET levels have been observed in various cancers; however, the underlying molecular mechanisms remain unclear. We found that SET protein accumulates with the increasing density of cultured cells. In this study, we aimed to clarify the mechanism underlying the accumulation of SET.

High cell density increased the SET levels in all the adherent cells analyzed. This phenomenon was observed in both cancer cell lines and non-cancer cell lines. The mRNA levels of SET were not affected by cell density, while the half-life of SET was extended at high cell densities. Autophagy inhibition led to SET accumulation, indicating the involvement of autophagy. However, cell density does not affect global autophagy activity, suggesting the involvement of selective autophagy in SET degradation. SETBP1 directly binds to SET and protects it from cleavage by proteases. We found that high cell density increased SETBP1 mRNA and protein. Furthermore, altering the expression of SETBP1 suppressed the change in SET with cell density. Our data revealed a mechanism underlying the regulation of SET level, wherein increased cell density induces SETBP1 expression and protects SET from selective autophagy.

Methyl vinyl ketone (MVK) is an α,β-unsaturated carbonyl compound contained in smoke and exhaust gas. This compound is known to covalently bind to proteins via Michael reaction. However, the target proteins are fully unsolved yet. We found that MVK suppresses phosphatidylinositol 3-kinase (PI3K) –Akt signaling. This pathway is essential for various biological regulation, including cell survival, glucose metabolism, and autophagy. In this study, we tried to clarify the mechanism by which MVK interferes with PI3K–Akt signaling.

Initially, we investigated the effects of MVK on the phosphorylation of epidermal growth factor receptor (EGFR), PI3K and Akt by treatment with EGF in A549 cells. MVK significantly attenuated the levels of pAkt and pPI3K, but not pEGFR formation. In addition, co-immunoprecipitation analysis revealed that MVK inhibits the interaction of PI3K with EGFR. Interestingly, exposure to MVK did not change the levels of phosphatidylinositol 3,4,5-trisphosphate indicating that the catalytic domain in PI3K is not a target of MVK. Next, we employed LC-MS/MS analysis to determine the modification sites in PI3K. We identified that both Cys146 and 656 residues in PI3K p85 subunit are modified with MVK. To confirm if these sites are essential for the EGF signaling, we substituted each Cys residue with Ser. We found that EGF treatment fails to activate the phosphorylation of PI3K p85 C656S mutant but not the C146S mutant. These results indicated that MVK may be a novel type of PI3K inhibitor via modification of p85 subunit.

Epigenetic dysregulation, such as aberrant DNA methylation, is one of the hallmarks of cancer cells, however the mechanisms by which these dysregulations occur remain unclear. Previously, we found that DNA methyltransferase 3B (DNMT3B), which catalyzes cytosine methylation, is selectively oxidized by several stresses. A cysteine residue is the modification site on DNMT3B. As oxidation of DNMT3B attenuates its enzymatic activity, this reaction may be related to the epigenetic regulation. In this study, we focused on the cell cycle regulator CCND2 and examined whether its expression is regulated by oxidative stress and enzymatic activity of DNMT3B. Indeed, exposure to the stress and the universal DNMT inhibitor 5-aza-2'-deoxycytidine induced CCND2 expression. When DNMT1, DNMT3A, and DNMT3B were overexpressed, only DNMT3B significantly reduced CCND2 mRNA levels. CS mutant, which lost enzymatic activity, had no effect on CCND2 mRNA levels compared with the wild type. The siRNA knockdown of DNMT3B markedly enhanced CCND2 mRNA levels. Our data strongly suggest DNMT3B-specific regulation of CCND2 expression. Finally, bisulfite sequencing analysis revealed that oxidative stress significantly decreased DNA methylation levels in the CCND2 promoter region. These results indicate that oxidation of DNMT3B plays an important role in epigenetic regulation and may lead us to know the mechanism of epigenetic dysregulation in cancer cells.

Differentiation-inducing factor 1 (DIF-1) is a polyketide produced by slime mold Dictyostelium discoidium which inhibits growth and migration and promotes differentiation of Dictyostelium cells by localizing to mitochondria. DIF-1 regulates phosphorylation of signaling molecules involved in rearrangement of actin cytoskeleton and induces transient cortical accumulation of F-actin in Dictyostelium cells. We recently reported that DIF-1 inhibits growth and migration of various types of mammalian cell lines through, at least in part, activation of AMP-activated kinase (AMPK). However, the molecular mechanism for the effect of DIF-1 on actin dynamics remains elusive. Here, we found that DIF-1 regulates actin cytoskeleton in endothelial cells by activating Cofilin, an actin depolymerization factor. In mouse immortalized endothelial cells (SVECs), DIF-1 rapidly induced dephosphorylation and activation of cofilin, followed by actin fiber depolymerization. When Cofilin is activated, mitochondrial fission is induced. Consistently, DIF-1 induced mitochondrial fission and knockdown of Cofilin suppressed mitochondrial fission by DIF-1. These results suggested that DIF-1 regulates actin cytoskeleton and induces mitochondrial fission via Cofilin activation.

【Purpose】

COPD is a refractory pulmonary disease characterized by bronchitis, emphysema and mucus stasis. Because COPD is a multifactorial disease and represents various symptoms for each patient, previous studies which regard COPD as a single disease have limits. Therefore, to establish personalized medicine for COPD we need research developments based on phenotype or endotype classification. Here, I focused on T₃ which has long been known to act as an essential factor for development and growth in the lung. This study aims to elucidate the pathophysiological role of T₃ using COPD mouse model simulating disease type classification (emphysema- or airway-dominant). 

【Methods & results】

In this study, I chose elastase-induced model and C57BL/6-βENaC-Tg mice as mouse models of emphysema- and airway-dominant COPD, respectively. I intratracheally administered T₃ (40 or 80 μg/kg, every other day) to elastase-induced mice for 21 days or C57BL/6-βENaC-Tg mice for 12 days. In elastase-induced model, T₃ treatment improved emphysema and, partially, the respiratory function with upregulation of the expression of Ppargc1a (the master regulator of mitochondrial biogenesis) and Gclm (an oxidative stress-related factor) after one T₃ injection. On the other hand, in C57BL/6-βENaC-Tg mice T₃ treatment did not improve COPD pathology.

【Discussion】

These results emphasize the importance of research developments based on phenotype or endotype classification considering that T₃ effect is different between two COPD models.

Fluoro-loxoprofen (F-LOX), which is a derivative of loxoprofen (LOX), has been reported to be less ulcerogenic response to the gastric mucosa, but its effect on the small intestinal mucosa has not been investigated. In this study, we investigated the influence of the novel anti-inflammatory drug F-LOX on the small intestine in rats.

F-LOX and LOX were orally administered to male SD rats, and the total area of macroscopic injury in the small intestine was determined 24 h later. The prostaglandin E2 (PGE2) production and cAMP contents in the small intestine were examined 3 or 6 h after administration of drugs by enzyme immunoassay.

LOX caused hemorrhagic injury along the small intestine, mainly in the jejunum and ileum 24 h later. The intestinal lesion was also observed in the F-LOX group, but it was less, and the severity was lower than in the LOX group. The PGE2 production in the small intestine was significantly decreased 3 h after LOX and F-LOX administration. The cAMP contents were significantly reduced in the LOX group but not in F-LOX group. A decrease in PAS-positive mucus was observed in the F-LOX group, but the amount of mucus was higher than that in the LOX group. In conclusion, F-LOX is an NSAID that has the same anti-inflammatory effect as LOX but is less damaging to the small intestinal mucosa.

Although an antitumor drug 5-fluorouracil (5-FU) frequently causes intestinal mucositis accompanied by severe diarrhea, the useful prevention and treatment have not been established. Glutamate is known to play an important role in energy metabolism in gastrointestinal tracts. The aim of this study was to investigate the alleviative effect of 5-FU-induced mucositis in mice. Intestinal mucositis was induced in male C57BL/6 mice by repeated administration of 5-FU for 6 days. Glutamate was administered orally starting from 5 days before the onset of 5-FU treatment. Disease severity was assessed by body weight and stool consistency, and the intestinal mucositis was examined histologically. The effect of glutamate on 5-FU-induced cell injury was also examined in rat intestinal epithelial cell line IEC6.

Repeated administration of 5-FU produced severe intestinal mucositis, histologically characterized by the shortening of villi and destruction of crypts, accompanied by body weight loss and diarrhea. Daily administration of glutamate significantly reduced the severity of histological intestinal injury despite little preventive effect on diarrhea and body weight loss. The pretreatment with glutamate significantly increased epithelial electrical resistance in IEC6 cells. These results suggest that glutamate prevents 5-FU-induced intestinal mucositis via enhancement of intestinal barrier functions. Thus, glutamate administration may be useful for prevention and treatment of intestinal mucositis during cancer chemotherapy.

Cisplatin-induced ototoxicity (CIO) is caused by cisplatin accumulation in the inner ear cochlea, which is mediated by organic cation transporter 2 (OCT2). Proton pump inhibitors, including lansoprazole (LPZ), ameliorated cisplatin-induced nephrotoxicity via inhibiting OCT2. In the present study, we investigated the protective effect of LPZ against CIO using zebrafish and real-world data. Using zebrafish, we compared the effect of LPZ on CIO through in vivo fluorescence imaging of the hair cells stained with fluorescence dyes. Cisplatin treatment to zebrafish significantly decreased the fluorescence intensities (FI) of hair cells (approximately 50% of those of control zebrafish). Co-treatment of LPZ or knockout (KO) of oct2 significantly suppressed the reduction of FI by cisplatin (approximately 75% of those of control zebrafish). The protective effect of LPZ were not significantly different between wild type and oct2-KO zebrafish. Using electronic medical records in Mie University Hospital, we validated the preventive effect of LPZ against CIO in 289 patients who received cisplatin contained chemotherapy. The rate of co-administrated LPZ in patients without ototoxicity was significantly higher than that of patients with ototoxicity (34% vs 7%). These results suggest that LPZ should suppress CIO through the inhibition of OCT2.

Cancers evade immunity by multiple mechanisms. Myeloid-derived suppressor cells (MDSCs) have been shown to be involved in immunosuppression in cancer patients. They are suggested to be increased and activated by tumor microenvironment. Subpopulations of MDSCs have been shown to be monocytes/macrophage-lineage cell. In addition, subpopulations of tumor-associated macrophages have also been shown to suppress inflammation and immune response in tumors, suggesting that tumor microenvironment may educate monocytes/macrophages to acquire the immune regulatory function. To test this hypothesis, we first injected a mixture of bone marrow-derived macrophages (BMDMs) and LL/2 lung cancer cells subcutaneously into mice. We found that BMDMs increased tumor growth. To further analyze, we cocultured BMDMs with LL/2 cells. Interestingly, coculturing with LL/2 dramatically increased expression of Cd274, encoding an immune checkpoint protein PD-L1. They also increased Vegf (vascular endothelial growth factor) expression. These results suggest that LL/2-educated BMDMs may promote tumor growth. Indeed, LL/2-educated BMDMs suppressed activation of CD8⁺T cells, indicating they acquired immune suppressive activity. Our data support the notion that tumor microenvironment educate macrophages so that they support tumor survival and growth.

Ozone water is currently utilized for antibacterial and antiviral purposes under the pandemic of COVID-19 due to its strong oxidation effect without safety problems. Therefore, clinical applications of ozone water for cutaneous diseases such as atopic dermatitis (AD) and pyoderma are expected recently. The aim of this project is to apply the ozone water for AD and pyoderma in dogs. We here examined the antibacterial effects of ozone water using staphylococci in in vitro setting, and anti-allergic effects of ozone water in AD mouse model.

We first examined the bactericidal effect of ozone water on resident staphylococci on skin. Staphylococcus aureus, which is an exacerbating factor in human AD, and S. lentus collected from AD mouse model was treated with ozone water and the number of bacteria was determined. Our findings indicated that ozone water showed a significant bactericidal effect against both staphylococcal species . We are currently investigating the efficacy of ozone water against staphylococci isolated from AD and pyoderma dogs. In vivo experiment with AD mouse model is also underway to look for the therapeutic effects of ozone water with impact on inflammatory and itch responses.

Tetranor-Prostaglandin D Metabolite (tetranor-PGDM) is a metabolite of PGD₂. Urinary tetranor-PGDM level is increased in some diseases, including food allergy. In this study, we developed a monoclonal antibody (MAb) and a competitive enzyme immunoassay (EIA) for measuring tetranor-PGDM. Spleen cells isolated from mice immunized with tetranor-PGDM were utilized to generate Ab-producing hybridomas. We chose hybridomas and purified MAb against tetranor-PGDM to develop competitive EIA. The assay evaluated the optimal ionic strength and pH. Specificity was determined by cross-reactivity to tetranor-PGEM, tetranor-PGFM, and tetranor-PGAM. Recovery was determined by spiking experiments on artificial urine. Optimal ionic strength was 150 mM NaCl, and optimal pH was pH 7.5. Metabolites other than tetranor-PGDM did not show any significant cross-reactivity in the EIA. The assay exhibited range of quantitation (ROQ) value of 0.252 to 20.2 ng/mL. The linearity-dilution effect showed excellent linearity under dilution when artificial urine samples were applied to solid-phase extraction (SPE). After SPE, recovery of tetranor-PGDM in artificial urine averaged from 82.3% to 113.5% and was within acceptable limits (80%–120%). We successfully generated one monoclonal antibody and developed a sensitive competitive EIA.

【Introduction】Chronic myelogenous leukemia (CML) has become a disease with five-year-survival rate of more than 90% according to appearance of Bcr-Abl inhibitor. Stopping treatment with Bcr-Abl inhibitors leads to relapse in many patients. This is due to the fact that Bcr-Abl inhibitors are not effective against cancer stem cells, and suggests that a different drug is necessary to eradicate cancer stem cells. ALDH is overexpressed in cancer stem cells and promotes their survival. We have shown in past studies that Bcr-Abl inhibitors do not inhibit ALDH expression. In this study, we examine the effect of ALDH inhibitors on CML.

【Methods】We examined the effects of an ALDH inhibitor using K562 cells, which are CML cell line. We verified the effect of an ALDH inhibitor against CML using a WST-8 assay. We then measured ALDH protein expression using flow cytometry.

【Results】ALDH inhibitors reduced cell survival in a concentration-dependent manner, and when combined with a Bcr-Abl inhibitor, cell viability was synergistically decreased. ALDH protein expression was decreased in cells treated with ALDH inhibitor alone or Bcr-Abl inhibitor alone according to flow cytometry. In cells treated with ALDH inhibitor and Bcr-Abl inhibitor, ALDH protein expression was more strongly repressed.

【Conclusion】The results of this study suggest that ALDH inhibitor use might be effective in CML treatment.

Fatty acid coenzyme A (CoA), which is synthesized from fatty acids and CoA, plays an important role in lipid metabolism as it is used for beta oxidation and lipid biosynthesis. Beta oxidation, a metabolic pathway for degrading fatty acid CoA to extract acetyl CoA, occurs at mitochondria and peroxisomes. Beta oxidation in mitochondria plays a vital role in supplying acetyl CoA, FADH2, and NADH to TCA cycle, whereas in peroxisomes plays an important role in the degradation of lipids, including very long chain fatty acid CoA, which cannot be transported to mitochondria. ABCD1, ABCD2, and ABCD3, which belong to the ATP-binding cassette transporters (ABC transporters), transport long chain fatty acid acyl CoA from cytoplasm to peroxisome. ABCD1, 2, and 3 have a difference in the specificity of substrate. ABCD1 and 2 mainly transport saturated long chain fatty acid CoA, whereas ABCD3 transports unsaturated long chain fatty acid CoA and branched fatty acid CoA. Several structures have been reported about ABCD1, but the structure of ABCD3 has not yet been elucidated. We analyzed the structure of ABCD3 to obtain a detailed structural basis for its substrate specificity. As a result, we determined ATP bound ABCD3 structure reconstituted into lipid nanodisc which is similar to natural lipid environment than detergent at 4.5 Å. In the near future, we will acquire the structure of ABCD3 bound to its substrate and will elucidate the molecular basis of substrate specificity among ABCD family, providing insights into diseases caused by abnormal lipid metabolism.