FPL64176 is a nondihydropyridine agonist of Cav1.2 Ca²⁺ channels. Although it is very potent and efficacious, the molecular mechanism underlying its action has not been unequivocally identified. Because it was previously proposed that it may augment the channel currents by making inactivated channels permeable, we here examined its effect on recombinant Cav1.2 Ca²⁺ channels (alpha_1C/beta_2a/alpha₂delta₁) bearing mutations impairing different types of inactivation. Voltage-dependent inactivation (VDI) caused by a cytoplasmic linker between domains I and II of alpha_1C subunits was inhibited by a mutation causing Timothy syndrome 1, whereas VDI caused by a distal C-terminus (DCT) of alpha_1C subunits was inhibited by deletion of DCT. Ca²⁺-induced inactivation (CDI) caused by the binding of Ca²⁺ to calmodulin (CaM) preassociated with an IQ domain in the proximal C-terminus (PCT) of alpha_1C subunits was inhibited by coexpression of dominant negative CaM. We found that FPL64176 augmented the channel currents depending on CaM-mediated CDI and DCT-mediated VDI. It is known that the EF-hand in PCT is involved in both these types of inactivation. Therefore, it is likely that FPL64176 allosterically modulates the regulation of EF-hand by DCT and PCT associated with Ca²⁺/CaM.

Pulmonary artery hypertension (PAH) is a rare, progressive, and fatal cardiovascular disease. Several epidemiological PAH registries showed that the most common age of PAH patients is between 41-50 years (mean age of 46.8 years). However, the reason why PAH is more susceptible for young remains unclear. In the present study, the survival rate and morphological changes of monocrotaline (MCT)-induced pulmonary hypertensive (PH) rats were compared between young (4-week-old) and adult (17-week-old). The Kaplan-Meier survival analysis revealed that young sham-treated rats were survived at day 35, whereas young MCT-PH rats were died at mean 22.4 days after MCT injection. Interestedly, adult MCT-PH rats were survived over 42 days, similar to adult sham-treated rats. The weights of heart and pulmonary artery were increased in young MCT-PH rats. Similar increases were also observed in adult MCT-PH rats. In addition, the expression level of Ca²⁺-sensing receptor (CaSR) and transient receptor potential canonical channel 6 (TRPC6), which were identified as molecules associated with the pathogenesis of PAH, were compared between young and adult MCT-PH rats. Their expressions were upregulated in both groups. Further analyses are necessary for elucidating the mechanism underlying the age difference in PAH patients.

Ca²⁺-activated K⁺ channel K_Ca1.1 has a potential as a prognostic tumor marker in several solid cancers. Three-dimensional (3D) in vitro cell culture system mimics in vivo solid tumors resistance to chemotherapy in tumor microenvironment (TME). In the MG-63 cells isolated from 3D spheroid models, K_Ca1.1 activator-induced hyperpolarizing responses were largely enhanced, compared with adherent 2D monolayer cells. In lipid-raft-enriched compartments of MG-63 spheroids, the protein expression level of K_Ca1.1 was significantly increased, without changing its transcriptional level. The spheroid formation caused down-regulation of the ubiquitin E3 ligase FBXW7, and its inhibition in 2D monolayer cells increased the K_Ca1.1 protein expression. In the MG-63 spheroids, treatment with the K_Ca1.1 inhibitor suppressed chemoresistance ability to paclitaxel, doxorubicin, and cisplatin. Of several multidrug resistance ABC transporters, a multidrug resistance-associated protein, MRP1 was up-regulated in the MG-63 spheroids, and chemosensitivity was recovered by the K_Ca1.1 inhibition. Taken together, pharmacological inhibition of K_Ca1.1 may be an attractive new strategy for conquest of chemotherapeutics synergizing in the hypoxic TME of K_Ca1.1-positive solid cancers.

Several hERG blockers with good clinical safety profiles share a common attribute: They all paradoxically increase hERG current at potentials near the threshold for its activation. We refer to this phenomenon as "facilitation." The molecular mechanism underlying facilitation remains unknown. Here, we propose a mechanism through which facilitation arises from drug effects on hERG channel gating kinetics. We found that 1) nifekalant, a hERG facilitator, accesses the receptor site within the pore of the open or inactivated channels at depolarized potentials, 2) upon return to the resting potentials, channels close trapping nifekalant inside, 3) trapped nifekalant biases the open-closed equilibrium towards the open state, and 4) the kinetics of drug escape from the channel are faster than channel closing rates at potentials where facilitation of hERG current is observed, thereby drug unbinding reveals channels that have been biased towards the open state. Simulations with a Markov model of such nifekalant-hERG interaction successfully reproduce key characteristics of hERG facilitation. Therefore, hERG facilitation arises from the coupling of the features of the channel gating, slow opening and closing, with a drug action on the gating, similar to a classic "foot-in-the-door" mechanism.

In immune cells, the activation of K⁺ channels causes membrane hyperpolarization and Ca²⁺ increase through voltage-independent Ca²⁺ channels, thus controls various cellular functions. The inward rectifier K⁺ channel Kir2.1 has been reported to be functionally expressed in leukocytes, but its physiological significance is unclear. This study was undertaken to elucidate the physiological roles of Kir2.1 channels in macrophage functions using mouse bone marrow-derived macrophages (BMDM). Whole-cell patch-clamp recordings revealed that Kir2.1 channels were responsible for inward rectifier K⁺ currents in BMDM. Membrane hyperpolarization by the activation of Kir2.1 channels promoted store-operated Ca²⁺ entry (SOCE) through Ca²⁺ release-activated Ca²⁺ (CRAC) channels in BMDM treated with thapsigargin or ATP. Migration experiments by scratch assay demonstrated that the activities of both Kir2.1 and CRAC channels enhanced cell migration. In addition, the cell migration was significantly inhibited by an inhibitor of Ca²⁺/calmodulin-dependent protein kinases (CaMK). These results suggest that Kir2.1 channels activate CaMK and promote cell migration by increasing SOCE through CRAC channels in BMDM. Kir2.1 channels may enable efficient migration of macrophages to damaged or/and inflamed sites and be involved in cell defense mechanisms.

Store-operated Ca²⁺ entry (SOCE) plays critical roles in intracellular Ca²⁺ ([Ca²⁺]_i) homeostasis. Recent studies have shown that SOCE is essential for osteoblastic differentiation. In non-excitable cells, K⁺ channels are key regulators of SOCE-mediated Ca²⁺ signaling and control cell proliferation, differentiation, and migration, however, the functional role of K⁺ channels in osteoblast Ca²⁺ signaling remains unknown. In the present study, the contribution of K⁺ channels to the SOCE activity in the osteoblastic cell line MC3T3-E1, established from mouse calvaria was investigated. We found that the expression levels of inward rectifier K⁺ channel Kir2.1 transcripts and proteins were up-regulated in the differentiated MC3T3-E1 cells. The application of ML133, a Kir2 inhibitor, significantly reduced the SOCE-mediated [Ca²⁺]_i elevation in differentiated MC3T3-E1 cells, and suppressed the expression of the differentiation markers in osteoblasts. In addition, the expression levels of Kir2.1 proteins were up-regulated in murine embryonic metatarsals, depending on the progression of the endochondral ossification. These results suggest that Kir2.1 channels play essential roles in maintaining the bone homeostasis via modulating osteoblast differentiation.

Activated phonotype of hepatic stellate cells is associated with the development of liver fibrosis following enhanced Ca²⁺ signaling. Although cytosolic Ca²⁺ concentration is regulated by Ca²⁺ and K⁺ channels, their pathophysiological roles remain unclear. In the present study, we focused on the two-pore domain K⁺ (K_2P) channels, which regulate the resting membrane potentials, in hepatic stellate cells. In the presence of blockers for voltage-dependent K⁺ channels (10 mM tetraethylammonium and 5 mM 4-aminopyridine) and small-conductance Ca²⁺-activated K⁺ channels (100 nM apamin), outward K⁺ currents were detected in human hepatic stellate LX-2 cells. Expression analyses revealed that, among the K_2P family, TREK-1 (KCNK2) channels were abundantly expressed in LX-2 cells. TREK-1 siRNA (20 nM, for 48~72 h) specifically knocked-down the expression of TREK-1 channels. TREK-1 siRNA did not affect the expression of α-SMA (a marker for cell motility). On the other hand, TREK-1 siRNA reduced the expression of type I collagen (an extracellular matrix). TREK-1 siRNA also downregulated the expression of platelet-derived growth factor-BB (a cytokine associated with cell proliferation). These results suggest that TREK-1 channels contribute to the extracellular matrix production and cell proliferation in hepatic stellate cells.

Melatonin is secreted from the pineal glands and regulates the circadian rhythms. It has been reported that melatonin modulates the activity of ion channels. In the present study, the effects of melatonin on voltage-dependent K⁺ (K_V) channels, which contribute to the regulation of resting membrane potentials and action potentials, were examined in rat pinealocytes and reconstituted HEK293 cells. The expression analysis by quantitative real-time PCR revealed that K_V4.2 channels was abundantly expressed in rat pineal glands. In rat pinealocyte, 5 mM 4-aminopyridine-sensitive K_V4.2 currents were detected using whole-cell patch-clamp configuration. In HEK293 cells expressed with K_V4.2 channels, the application of melatonin (100 µM, 300 µM, and 1 mM) inhibited K_V4.2 currents in a concentration-dependent manner. The inhibitory effects of melatonin on K_V4.2 currents were observed even in the presence of 100 nM luzindole, an inhibitor of the melatonin receptors. Neither 1 mM tryptophan nor 1 mM serotonin, which are the precursors to melatonin, affected the activity of K_V4.2 currents. These results strongly suggest that melatonin inhibits K_V4.2 channels directly, which may contribute to the regulation of physiological functions in pineal glands.

Major depressive disorder is one of the largest medical and social problems worldwide. 

Previous studies have shown that the activity of serotonin neurons in the dorsal raphe nucleus (DRN) is increased by chronic administration of a selective serotonin reuptake inhibitor (SSRI) and decreased by chronic social defeat stress (CSDS), a mouse model of depression. These reports have indicated the pivotal role of DRN serotonin neurons in depression, although the underlying mechanism is still unclear. To elucidate the mechanism, we have applied the Translating Ribosome Affinity Purification (TRAP) technique to isolate the mRNA of the DRN serotonin neurons. First, we tried to confirm that TRAP can enrich the serotonin-neuron-specific mRNA and deplete the mRNA of other cell types. Microarray analysis revealed that marker genes of serotonin neurons such as Tph2, Ddc, and Slc6a4 were enriched in immunoprecipitated (IP) samples compared to input, whereas glial marker genes such as Mbp and Gfap were depleted in IP sample. Then we performed TRAP of the DRN serotonin neurons from naïve, SSRI-treated mice, as well as resilient and susceptible mice after CSDS. Differential expression analysis identified 297 genes whose expression was increased by chronic SSRI treatment and decreased by CSDS, and 217 genes was increased by CSDS and decreased by SSRI. Identification of the causal link between the differentially expressed genes and the behavioral changes will help not only to clarify the pathophysiology of depression but also to provide therapeutic targets for drug discovery.

It has been shown that activation of the glutamatergic transmission in the prelimbic region of the mPFC (PL-PFC) evoked anxiety-like behavior in rodents. We previously reported that local perfusion of a selective agonist to delta-opioid receptor (DOP), KNT-127, attenuated the veratrine-induced elevation of extracellular glutamate in the PL-PFC and anxiety-like behavior in mice. These results suggested the possibility that KNT-127 suppresses glutamate release from presynaptic site in the PL-PFC. To confirm this, we performed whole-cell patch-clamp recording from pyramidal neurons in the PL-PFC, and examined the spontaneous and electrically-evoked excitatory post-synaptic currents (EPSC)s. We found that bath application of KNT-127 significantly suppressed frequency, but not amplitude of spontaneous and miniature EPSCs in a DOP-dependent manner. Also, KNT-127 increased paired-pulse ratios in the PL-PFC pyramidal neurons tested. Further, we analysed the firing properties of pyramidal neurons in the PL-PFC, and found that KNT-127 treatment significantly reduced the number of action potentials and rheobase. These results suggested that KNT-127 not only suppresses glutamatergic synaptic transmission by inhibiting glutamate release from presynaptic site, but also reduces cell excitability via DOP in the mouse PL-PFC.

Indoleamine 2,3-dioxygenase 1 (IDO1) is the first rate-limiting enzyme that metabolizes tryptophan to the kynurenine pathway. Its activity is highly inducible by pro-inflammatory cytokines and correlates with the severity of major depressive disorder (MDD). MicroRNAs (miRNAs) are involved in gene regulation and the development of neuropsychiatric disorders including MDD. However, the role of miRNAs in targeting IDO1 in the pathophysiology of MDD is still unknown. In the present study, we investigated the role of novel miRNAs in the regulation of IDO1 activity and its effect on lipopolysaccharide (LPS)-induced depression-like behavior in mice. LPS upregulated miR-874-3p concomitantly with increase of IDO1 expression in the prefrontal cortex (PFC), increase of immobility in the forced swimming test as depression-like behavior and decrease of locomotor activity as sickness behavior without motor dysfunction. The miR-874-3p increased in both neuron and microglia after LPS. Its mimic significantly suppressed LPS-induced IDO1 expression in the PFC. Infusion of IDO1 inhibitor (1-methyl-l-tryptophan) and miR-874-3p into PFC prevented an increase of immobility in the forced swimming test, but did not decrease of locomotor activity induced by LPS. These results suggest that miR-874-3p may play an important role in preventing the LPS-induced depression-like behavior through inhibition of IDO1 expression. This may also serve as a novel potential target molecule for the treatment of MDD.

Major depressive disorder (MDD) is a common mental disorder characterized by reduced motivation, diminished interest and pleasure, and anhedonia. We have proposed melanoma-associated antigen D1 (MAGE-D1) knock out (KO) mouse is a MDD model, and which involves the serotonergic hypofunction. However, not only serotonergic but also noradrenergic neuronal malfunctions are involved in depressive behaviors. Here, we investigate the involvement of noradrenergic neuronal system in depression-like behaviors of MAGE-D1 KO mice. MAGE-D1 KO mice showed decreases in locomotor activity, social interaction time and sucrose preference, but increases in immobility time in the forced swimming test (FST), and feeding latency in the novelty suppression feeding test. Noradrenaline (NA) tissue contents in the prefrontal cortex, hippocampus, and amygdala, and potassium-evoked noradrenaline releases in the prefrontal cortex and hippocampus were decreased in MAGE-D1 KO mice. The protein expression of noradrenaline transporter (NAT) was increased in the prefrontal cortex of the MAGE-D1 KO mice. Phosphorylation of NAT at threonine and protein expression of its kinase, protein kinase C (PKC) were decreased, but not changed in ubiquitination or expression of NAT mRNA. Acute administration of NA reuptake inhibitors (desipramine and atomoxetine) attenuated increase in immobility time in the FST and decrease in sucrose preference, but not other behavior changes in MAGE-D1 KO mice. These results suggested that depression-like behaviors in MAGE-D1 KO mice might be associated with hypofunction of noradrenergic neuronal system due to NAT overexpression through decrease in PKC-dependent phosphorylation of NAT.

Major depressive disorder (MDD) is a worldwide serious psychiatric disease, and more than 300 million people suffer from MDD. Chronic stress contributes to the pathogenesis of MDD. Cigarette smoking is strongly associated with MDD. Epidemiological and clinical studies claim that the smoking is assumed as self-medication for stress and depression. In this study, we investigated the effect of nicotine on the depression-like behavior induced by chronic unpredictable mild stress (CUMS). At the age of 6 weeks, C57BL6J mice were randomly exposed to 9 kinds of mild stressors for 4 weeks. Nicotine (0.2mg/kg), galantamine (1mg/kg) and varenicline (1mg/kg) were administrated 30 min before exposure to each stressor during CUMS. After CUMS, mice were subjected social interaction test and measured serum corticosterone levels and mRNA levels of  nicotinic acetylcholine receptor (nAChR) subunits in the prefrontal cortex (PFC). Nicotine attenuated decrease in social interaction time of CUMS mice. Nicotine did not affect elevated serum corticosterone levels immediately after CUMS but reversed the sustained elevation after behavioral test. CUMS did not affect mRNA levels of α7, α4 or β2 nAChR subunit in the PFC. Finally, we evaluated the efficacies of galantamine, α7 nAChR allosteric modulator and varenicline, α4β2 nAChR partial agonist on CUMS mice. Administration of galantamine, but not varenicline attenuated decrease in social interaction time of CUMS mice. These data suggested that α7 nAChR is an important target for stress resilience and development of antidepressant.

White matter abnormalities have been implicated in psychiatric diseases such as major depressive disorder (MDD) ; however, the underlying mechanisms remain poorly understood. The structure and function of the corpus callosum are particularly vulnerable to stress, which may lead to MDD. In the present study, we investigated whether chronic social defeat stress (CSDS) induces myelin abnormalities of the corpus callosum through inflammation that contributes to the pathogenesis of MDD. To produce CSDS, the adult C57BL/6J mouse was exposed to an aggressor ICR mouse for 10 consecutive days. CSDS decreased mature oligodendrocytes in the corpus callosum, and persistently developed depression-like behaviors such as increased immobility in the forced swimming test and impaired social interaction. On transmission electron microscopy, myelin abnormalities and axonal degeneration were observed with necrosis-like cell death of oligodendrocytes in the corpus callosum. Interestingly, CSDS significantly increased the Gasdermin D (Gsdmd), a marker of pyroptosis, concomitantly with enhanced IL-1β production in the corpus callosum. Administration of IL-1β inhibitor prevented the decrease of oligodendrocytes and CSDS-induced depression-like behaviors. These findings suggest that IL-1β acts as a crucial mediator of oligodendroglial pyroptosis induced by the CSDS, which may be responsible for the development of MDD.

Neuroinflammation is crucial for chronic stress-induced emotional disturbances. Stress-induced microglial activation occurs in selective brain regions (e.g., medial prefrontal cortex (mPFC)), but not in the others (e.g., nucleus accumbens (NAc)) and is sensitized with repetition of the stress. However, the mechanism remains unknown. Here we analyzed genome-wide patterns of social defeat stress-induced epigenetic and transcriptional changes in mPFC and NAc microglia. Before the stress, microglia in the two brain regions showed similar patterns of H3K27 acetylation in super-enhancers. The patterns diverged after the single stress and even more after the repeated stress. Some super-enhancers immediately responded to the stress specifically in the mPFC, whereas other super-enhancers gradually responded with repetition of the stress in both the two brain regions. These respective types of super-enhancers enriched different transcription factor binding motifs in their nucleosome-free regions. Furthermore, these epigenetic responses were concordant with the expression of adjacent genes associated with different biological functions. These findings show that stress induces multiple epigenetic regulations in microglia, which may contribute to the spatiotemporal patterns of neuroinflammation for emotional disturbances.

Chronic social stress affects mental and bodily functions and is a risk for depression and other stress-related disorders. Depression has been associated with increased leukocytes and reduced erythrocytes in the blood. Chronic stress in rodents recapitulates these blood profiles, and stress-induced leukocyte mobilization promotes depressive-like behaviors. However, the roles and mechanisms of chronic stress-induced anemia remain elusive. Here we found that chronic social stress reduced circulating iron levels and interfered with the iron-dependent erythrocyte maturation in the bone marrow, leading to iron-deficient anemia. However, systemic iron supplementation did not ameliorate this anemia but exacerbated concomitant depressive-like behaviors. These results demonstrate that chronic social stress alters iron metabolism, leading to anemia, yet promoting stress resilience.

Chronic early life stress (ELS) influences neuronal development and significantly increases the lifetime risk for the development of cognitive and affective pathology. It has been suggested that disruption in early life care impaired cognitive abilities in adulthood and affected neuronal development in the hippocampus in rodent models. However, the influences of ELS in emotional behaviors and hippocampal development in adolescence remain unknown. To clear these points, we examined the effects of ELS by housing mouse dam with limited nesting and bedding material from postnatal days 2-9 (P2-P9) and examined behaviors in 30 days (P30) of offspring. We also investigated hippocampal differentiation and microglia activation. Adolescent (P30) ELS mice showed over-response of stress escape in forced swim test. By immunohistochemical analysis, dentate gyrus of the hippocampus in ELS mice showed a significant increase in cell proliferation and survival. Furthermore, the number of IBA1 positive cells in the hilus was increased in stressed mice. Collectively, our findings demonstrate that chronic ELS affects adolescent behaviors and hippocampal development. It is necessary to investigate the relationship between these behaviors and hippocampal development in order to clarify the stress response in early childhood.

We have previously shown that various types of antidepressant treatments cause profound changes in maturation-related phenotypes of neurons in the hippocampal dentate gyrus (DG) of adult mice. Comprehensive gene analysis in the DG of antidepressant-treated mice, the expression of neurotrophin-3 (NT-3) mRNA is the most prominently reduced. In order to investigate the contribution of NT-3 reduction in the hippocampal function, NT-3 was knocked down specifically in the hippocampal dentate gyrus using an adeno-associated virus with artificial microRNA. Compared with the control group, hippocampal NT-3 KD mice showed dendrite elongation and increased density of immature neurons in the DG. It was also found that the expression of calbindin, a mature neuronal marker, was decreased in the granule cell layer of DG. Furthermore, the protein level of brain-derived neurotrophic factor (BDNF), which is known to increase with antidepressant treatment, increased in the DG. These results suggest that changes in hippocampal function due to NT-3 reduction are partly consistent with changes in hippocampal function due to antidepressant treatment.

The antidepressant actions of the NMDA receptor antagonist ketamine require activity-dependent release of brain-derived neurotrophic factor (BDNF) in the medial prefrontal cortex (mPFC). Our recent study shows a similar requirement for vascular endothelial growth factor (VEGF). Previous studies have reported that both BDNF and VEGF activate TRPC3 and TRPC6 via diacylglycerol production, and that activation of TRPC3 and TRPC6 mediates the neurotrophic effects of BDNF. Here, we examined the roles of TRPC3 and TRPC6 in the mPFC in the antidepressant actions of ketamine in lipopolysaccharide (LPS)-induced depression model mice. In both the tail suspension and forced swim tests, the antidepressant-like actions of ketamine were significantly blocked by intra-mPFC infusion of the selective TRPC3 inhibitor Pyr3 or the selective TRPC6 inhibitor SAR7334. Intra-mPFC infusion of BDNF or VEGF produced antidepressant-like effects, and these antidepressant-like effects were also blocked by intra-mPFC infusion of Pyr3 or SAR7334. Moreover, both systemic and intra-mPFC injection of a TRPC3/TRPC6 activator GSK1702934A produced antidepressant-like effects. These treatments did not significantly affect locomotor activity in LPS-treated mice. The current results indicate that the activation of TRPC3 and TRPC6 as downstream targets of BDNF and VEGF in the mPFC plays an essential role in the antidepressant actions of ketamine. TRPC3 and TRPC6 may be promising targets for the development of novel, rapid-acting antidepressant agents.

Our previous study demonstrated that the maintenance of hippocampal myelination contributes to the development of stress adaptation. Further, we showed that 5-HT_1A receptor in the hippocampus may be involved in the protection of major myelin proteins loss induced by unadaptable excessive stress. However, the mechanism by which activation of 5-HT_1A receptor promote myelination remains unknown. In the present study, we investigated the protective effects of 5-HT_1A receptor agonist flesinoxan on unadaptable stress-induced myelin damage and its molecular mechanisms in mice. Western blot analysis revealed that administration of flesinoxan significantly increased the expression level of BDNF, p-ERK, p-CREB, and Olig2 in the hippocampus of stress-maladaptive mice. Furthermore, in the immunohistochemical analysis, a significant increase in the number of Olig2⁺/BrdU⁺ positive cells were observed in the dentate gyrus of the hippocampus of flesinoxan-treated, but not vehicle-treated, stress-maladaptive mice. The present findings indicate that 5-HT_1A receptor-mediated enhancement of oligogenesis via activation of ERK/CREB/BDNF signaling pathways may be involved in the protection of myelin loss induced by unadaptable excessive stress.

Myelin sheath is essential for the rapid propagation of action potentials and proper functions of the nervous system. In the peripheral nervous system, it has been well-known that Schwann cells generate myelin sheath through several stepwise differentiation processes, but the detailed mechanism to regulate Schwann cell differentiation remains unclear. Previously, we demonstrated the involvement of hypoxia-inducible factor 1α (HIF1α) in the regulation of peripheral myelination. To further investigate the role of HIF1α in peripheral myelination, we used a conditional knock-out (cKO) mice lacking HIF1α gene in myelinating Schwann cells. An in vitro myelination assay revealed that hypoxic treatment facilitated myelination in wild-type control but not cKO. Unexpectedly, peripheral myelin of cKO mice was formed normally without significant motor deficits. Next, we examined the remyelination of cKO peripheral nerves after injury and found that the number of myelinated axons after nerve injury was decreased compared with that in control nerves. These findings suggest that HIF1α might be involved in peripheral remyelination after injury. To reveal the regulatory mechanism of HIF1α in peripheral myelination, we are performing  the comprehensive analysis of HIF1α regulated gene expression in Schwann cells.

Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder characterized by social deficit and stereotyped, repetitive patterns of behaviors and interests. Maternal use of valproic acid (VPA) during pregnancy is associated with an increased risk of ASD in the offspring. In the pathophysiological hypothesis of ASD, excitation/inhibition (E/I) imbalance is attracted. In this study, we investigated how VPA (500 mg/kg) at embryonic day 12.5 changes the emotional, cognitive and glutamatergic functions in the offspring of mice. Prenatal VPA exposure induced excessive repetitive self-grooming behavior, impairments of social behavior and object recognition memory, increased glutamatergic signaling [i.e. phospho-Ca²+/calmodulin-dependent protein kinase II (CaMKⅡ)and phospho-protein kinase C (PKC) levels] and decreased serotonin contents in the prefrontal cortex. These results suggested that VPA-exposure induced ASD-like behaviors associated with hyper-excitation of glutamatergic and hypo-serotonergic functions in the prefrontal cortex. Activation of serotonergic system by fluoxetine (20mg/kg) attenuated the VPA-induced ASD-like behaviors and hyper-glutamatergic signaling in the prefrontal cortex. These results suggest that hypo-serotonergic function is involved in the prenatal VPA-induced ASD-like behaviors and hyper-excitation in the prefrontal cortex.

We previously showed that the autism spectrum disorder (ASD) patient-derived de novo mutation (Q1038R mutation) in the Pogo transposable element with zinc finger domain (POGZ) protein is associated with ASD-like social behavioral deficits in mice. Considering its amino acid sequences and putative domain structures, POGZ is suggested to be involved in transcriptional regulation; however its transcriptional targets in the brain are largely unknown. In order to identify genes transcriptionally regulated by POGZ, we knocked down the expression of POGZ using shRNA method in the mouse primary neurons and performed RNA-sequencing for these neurons. We observed that the expression level of the oxytocin receptor (OXTR) gene is low in POGZ knocked-down neurons compared with control neurons. Interestingly, we found that the expression level of the OXTR gene is also low in the POGZ-Q1038R mutant mice, suggesting that the Q1038R mutation downregulates the function of POGZ. To examine the causal relationship between the decreased expression of the OXTR gene and ASD-like social deficits in the POGZ-Q1038R mutant mice, we intranasally administered oxytocin to the mice 30 minutes before the social interaction test. We found that intranasal oxytocin treatment effectively ameliorates the impaired social interaction in the POGZ-Q1038R mutant mice. These results suggest that the impaired social interaction may be caused by the altered oxytocin system in the POGZ-Q1038R mutant mice and provide insights into the development of treatment strategies of ASD.

There has been growing evidence suggesting disturbances in the neonatal period, including exposure to anesthetics and sedatives, cause neurodevelopmental disorders long after the causative event. In this study, we investigated the effect of neonatal treatment with midazolam, an allosteric agonist of gamma-Aminobutyric acid (GABA)_A receptor that bind to benzodiazepine receptor, on spontaneous activity and sociability in social recognition test in adulthood. In the social recognition test, rats were allowed to explore an open field where unfamiliar individual (to-be familiar individual) were presented (sample phase, 5 min), and after a delay of 30 min they were placed again in the same arena where familiar individual and novel individual were placed (test phase, 5 min). Midazolam (50 mg/kg) treatment from 5 to 7 days after birth increased social interaction duration and decreased social discrimination ratio, i.e. decreased preference to the novel individual in the social recognition test. Furthermore, acute systemic administration of methylphenidate (1.5 mg/kg), dopamine and noradrenaline reuptake inhibitor, 30 min prior to sample phase recover the decrement of discrimination ratio in the rats neontally treated with midazolam. Results suggest that neural circuits whose development are disrupted by neonatal midazolam treatment are associated with sociability, and at least partially modified by dopamine and noradrenaline.

Attention deficit/ hyperactivity disorder (AD/HD) is a mild developmental disorder (DSM-5). We reported that stroke-prone spontaneously hypertensive rat/Ezo (SHRSP/Ezo) had high validity as an AD/HD animal model, because of its behavioral phenotype. Recently, we revealed the NMDA receptor dysfunction on excitatory synapse in the prefrontal cortex (PFC) of SHRSP/Ezo. D-serine, an endogenous co-ligand for NMDA receptor, is one of new drug targets for the treatment of several psychiatric disorders. D-serine is biosynthesized from the optical conversion of L- to D- by serine racemase (SR), and metabolized by D-amino acid oxidase (DAAO). Here, we evaluated the serine kinetics in the PFC of SHRSP/Ezo and assessed the effect of DAAO inhibitor on AD/HD-like behaviors of SHRSP/Ezo.

At 6 weeks old, SHRSP/Ezo and its genetic control, WKY/Ezo were anesthetized and immediately decapitated. The PFC tissue was trimmed on ice for analysis of SR and DAAO expression by western blotting and measurement of serine concentration by HPLC-ECD system. Moreover, we performed local injection of DAAO inhibitor (5 microgram/side) into the PFC and assessed AD/HD-like behaviors using Y-maze test.

DAAO in the PFC of SHRSP/Ezo was significantly higher expression compared with WKY/Ezo, although SR was not significant. Correspondingly, D-/L- ratio of serine in the PFC of SHRSP/Ezo was lower than that of WKY/Ezo. Furthermore, local injection of DAAO inhibitor into the PFC improved inattention and hyperactivity of SHRSP/Ezo. These results suggest that DAAO inhibitor can be a possible candidate for treatment of AD/HD.

Obsessive-compulsive disorder (OCD) is a neuropsychiatric disorder characterized by repeated rising thoughts and repetitive unwanted behaviors. Selective serotonin reuptake inhibitors (SSRIs) are widely used as the first-choice treatment for OCD. Previously, we reported that mice received repeated injections of quinpirole (QNP; a dopamine D₂ receptor agonist; 1 mg/kg; 8 days) displayed OCD-like abnormalities, which were ameliorated by chronic SSRI administration. In this study, we aimed to investigate therapeutic mechanisms of SSRI for OCD. In acute brain slices from QNP-treated mice, chronic administration of an SSRI (citalopram; 24 mg/kg/day, 28 days) ameliorated hyperactivity of pyramidal neurons in the lateral orbitofrontal cortex (lOFC). SSRI administration increased the frequency of inhibitory inputs to lOFC pyramidal neurons. These effects of SSRI were mimicked by acute perfusion of SB242084 (a 5-HT_2c receptor antagonist; 3 μM). In contrast, SB242084 attenuated inhibitory inputs to lOFC fast-spiking interneurons, suggesting that 5-HT2c receptor antagonism inhibits lOFC pyramidal neurons by disinhibition of local fast-spiking interneurons. Consistent with electrophysiological experiments, short-term administration of SB242084 (1 mg/kg) improved reversal learning deficit in QNP-treated mice. These results revealed that chronic SSRI treatment improved OCD-like abnormalities through attenuation of 5-HT_2c receptor signaling in the lOFC, indicating a potential of 5-HT_2c receptor inhibition as a new therapeutic target for OCD.

Long-term use of dopamine D₂ receptor blockers (D2RBs) often causes tardive dyskinesia (TD), an adverse symptom characterized by involuntary hyperkinetic movements. Despite extensive research, there is no effective strategy to reduce the risk of TD. One approach for finding potential treatments of a drug-induced adverse events is to analyse clinical big data by searching hidden drug-drug interaction. Here, we analysed FDA Adverse Event Reporting System and JMDC insurance claims to determine an effective drug that lowers the incidence of D2RB-induced TD, and we found that acetaminophen decreased the risk of drug-induced dyskinesia in both clinical datasets. Experimentally, 21-day oral treatments of rats with haloperidol increased the number of vacuous chewing movements (VCMs), a parameter of TD in rodents, and this effect was inhibited by oral administration of acetaminophen. In mice, haloperidol-induced VCMs were also mitigated by oral administration of acetaminophen or intrastriatal injection of AM404, an acetaminophen metabolite that acts as a TRPV1 activator. Finally, acetaminophen mitigated the haloperidol-induced decrease in the number of c-Fos/preproenkephalin double-positive indirect-pathway medium spiny neurons of striatum in wild-type mice but not in TRPV1-deficient mice. These results suggest a new treatment for TD with high clinical predictability and a well-defined molecular mechanism.

Objective: To confirm the interaction between cholinergic and opioid systems in alcohol seeking behavior using an animal model. Methods: Experiments were conducted using Conditioned Place Preference(CPP) paradigm. Mice were conditioned with alcohol, nicotine and combination of both. They were then subjected to postconditioning test, in which their preference scores were measured. Following a period of drug abstinence, they were reinstated by morphine at doses of 5, 10, 20 and 40 mg/kg BW to induce relapse. Acetylcholinesterase (AChE) activity measurements were performed at the end of the behavioral tests using Ellman’s method. Results: Priming dose of morphine of10mg/kg, 20mg/kgand40mg/kg BW increased significantly the preference score during relapse to alcohol compared with the score in post conditioning test. AChE activity in animal at the time of relapse was significantly different compared to saline treated group. The highest enzyme activity was shown after priming dose of 20 mg/kg BW in group conditioned with alcohol and nicotine. There was no significant differences between the activity of AChE in groups receiving 5 mg/kg, 10 mg/kg, 20 mg/kg and 40 mg/kg BW of morphine challenge. Conclusion: Result of the present study indicated that morphine challenge in alcohol dependent animals during drug abstinence induces relapse to alcohol dependence. This is accompanied by increased AChE activity suggesting cholinergic-opioid system interaction

Alzheimer's disease (AD) is a neurodegenerative disorder that affects over 50 million people worldwide. Since it is known that the hypofunction of cholinergic system is one of pathologies associated with AD, acetylcholinesterase inhibitors (AChIs) such as donepezil are used for symptomatic treatment of AD; however, these drugs are broadly associated with increased risk for gastrointestinal adverse events, such as nausea and vomiting. Insufficient control of nausea and vomiting affects the ability to continue the AChIs therapy. We reported that pica, kaolin ingestion behavior, could be used to evaluate nausea and vomiting in mice. In this study, we investigated the effects of donepezil on pica in mice and the effects of anti-emetic drugs on inhibition of donepezil-induced pica.

Male mice were consecutively administered donepezil (5 mg/kg; i.p.) for 5 days, and their kaolin intakes were measured. Additionally, we examined the effects of a serotonin 5-HT₃ (granisetron: 0.5 or 1 mg/kg, i.p.), dopamine D₂ (metoclopramide 2.5 or 5 mg/kg; i.p.) and muscarinic (butylscopolamine 5 or 10 mg/kg: i.p.) receptor antagonists on the donepezil-induced pica. We found mice showed pica after 3 days of donepezil administration. Among the tested anti-emetic drugs, metoclopramide at the dose of 2.5 mg/kg significantly inhibited the pica in mice.

These results suggest donepezil-induced pica in mice has the potential to reflect donepezil-induced nausea and vomiting and dopaminergic pathway is involved in the development of in donepezil-induced nausea and vomiting human patients.

Tetrahydrobiopterin (BH4) is an essential cofactor for tryptophan hydroxylases, which is limiting step in serotonin (5-hydroxytryptamine, 5-HT) synthesis. We have previously reported the quinonoid dihydropteridine reductase (QDPR), BH4 regeneration enzymes, gene knockout (Qdpr^-/-) mice suppress platelet aggregation (92th congress). We further investigate its mechanism by 5-HT recovery using 5-hydroxytryptophan (5-HTP) supplement, precursor of 5-HT. Wild type (Qdpr^+/+) and Qdpr^-/- mice (OYC35, Lexicon Pharmaceuticals Inc.) were injected subcutaneously with 10 mg/kg of 5-HTP twice a day for 3 days. Intraplatelet 5-HT was quantified by high-performance liquid chromatography (HPLC) with electrochemical detection. Platelet aggregation were measured using platelet rich plasma (PRP), adjusted platelet count to 270,000/μL. The change in light transmission correspond to aggregation reaction was monitored after stimulation with either ADP or collagen. The intraplatelet 5-HT of Qdpr^-/- mice with 5-HTP supplement was recovered from 21.9±3.1 pmol/10⁶ platelets to 75.7±4.0, similar level of Qdpr^+/+ mice as 61.3±5.7. The area under the aggregation curve (AUC) of ADP-induced response was also recovered from 2,275±157 to 4,831±451 by 5-HTP administration correspond level to 5,436±696 of Qdpr^+/+ mice. The AUC of collagen-induced response revealed similar effects (Qdpr^+/+: 6,493±368, Qdpr^-/-: 4,402±589, Qdpr^-/-+5-HTP: 5,682±403). These results indicate that internal 5-HT disturbance is responsible for reduction of platelet aggregation in Qdpr^-/- mice.

We recently demonstrated that human aortic valve interstitial cells (HAVICs) obtained from patients with calcified aortic valve stenosis (AVS) were highly sensitive to ectopic calcification stimulation; however, the cell types contributing to calcification remains to be elucidated. We aimed to immunocytochemically characterize HAVICs and identify their contribution to valve calcification. HAVICs were isolated from patients with AVS and cultured on non-coated dishes. Immunocytochemical features and HAVIC differentiation were analyzed in passage 1 (P1). Most cultured P1 HAVICs were CD73-, CD90-, and CD105-positive, and CD45- and CD34-negative. HAVICs expressed vascular endothelial growth factor receptor 2 (VEGFR2); however, approximately 50% of the cells were α-smooth muscle actin (SMA)-positive, colonized, and easily differentiated into osteoblastic cells. Furthermore, VEGFR2-positive cells were more sensitive to tumor necrosis factor-α-induced ectopic calcification regardless of α-SMA expression. We concluded that HAVICs obtained from patients with calcified AVS are VEGFR2-positive undifferentiated mesenchymal cells and may contribute to aortic valve ectopic calcification.

Objectives:

Many cancer patients worldwide are treated with chemotherapeutics that cause various side effects, including erectile dysfunction (ED). We investigated the erectile function after the administration of the anti-cancer agent oxaliplatin (L-OHP), by using an animal model.

Methods:

Twelve-week-old male Wistar-ST rats were stratified into control and L-OHP groups. Rats were administered L-OHP (4 mg/kg i.v. 2 times/week for 4 weeks) in L-OHP group. Erectile and endothelial functions were measured using intracavernosal pressure (ICP) and isometric tension, respectively, after 4 weeks of L-OHP administration. Western blot analysis was used to assess the expression of neuronal nitric oxide (nNOS) and endothelial NO synthase (eNOS). Quantitative PCR was used to assess the expression of inflammation and oxidative stress markers (NADPH oxidase-1, p22phox, IL-6, and NF-κB).

Results:

The ICP/MAP ratio was significantly lower in the L-OHP group (P < 0.05). L-OHP group exhibited significantly lower responses to ACh, and nNOS and eNOS protein expressions and levels of inflammatory biomarkers were significantly higher in L-OHP group.

Conclusion:

L-OHP would cause ED through NO bioavailability reduction caused by not only neuronal dysfunction but also endothelial dysfunction. Therefore, cancer survivors who are administered L-OHP should be carefully screened for ED.

[Background]

Experimental autoimmune inflammation models have contributed to the understanding of pathogenesis of autoimmune diseases such as myocarditis and arthritis. In this study, we aim to establish an experimental autoimmune vasculitis (EAV) model.

[Method and Result]

Autoantibody against peroxiredoxin2(PRDX2) was reported to be detected in the serum of patients with Takayasu’s arteritis. Therefore, 8-week-old male Balb/c mice were immunized with recombinant mouse PRDX2 in complete Freund’s adjuvant twice on the 0th and 7th days (EAV0w and 1w). RT-qPCR analysis demonstrated that the expression of serum amyloid A-1/2(SAA-1/2) mRNA was increased in liver on the 21th day (EAV3w), suggesting that immunization with PRDX2 evoked systemic inflammation. Next, based on the database, we designed specific peptides which MHC classⅡ binds with, designated as PRDX2_79-98/_137-164. We immunized Balb/c mice with PRDX2_79-98/_137-164 peptides in the same manner, as above, and measured mRNA level. As a result, hepatic expression of SAA-1/2 mRNA was upregulated in EAV3w. Immunohistchemical analysis revealed that CD45 positive cells infiltrated around blood vessels in heart.

[Conclusion]

We have established an EAV model by immunizing PRDX2 peptides for the first time. Our EAV model could be beneficial for understanding of pathogenesis of vasculitis.

Recent studies have indicated that endothelial cell increases cellular stiffness upon inflammation and that stiff endothelium of atherosclerotic region and aged vessels is favor to monocyte adhesion and accumulation. The anti-coagulant recombinant soluble thrombomodulin (rsTM) suppresses inflammatory response of endothelial cells (ECs), however effect of rsTM on endothelial cellular stiffness remain elusive.  In this study, we investigated the impact of rsTM on lipopolysaccharide (LPS)-induced endothelial cellular stiffening.

We employed atomic force microscopy to measure the stiffness of cultured human umbilical vein ECs after LPS stimulation and/or rsTM treatment.  ECs increased their stiffness after 4 hours of stimulation, and rsTM reduced LPS-induced cellular stiffening through the attenuation of actin fiber and focal adhesion formation and via the improvement of gap junction functionality. LPS-induced ECs stiffening facilitated THP-1 cell adhesion, whereas rsTM suppressed THP-1 cell adhesion by reducing cellular stiffness.

Our finding that rsTM regulates monocyte adhesion via reducing endothelial cellular stiffness suggests that rsTM treatment holds some promise for the treatment of vascular inflammatory diseases, such as sepsis.

【Background】

p53 is reported to play important roles in the onset and progression of heart failure (HF) after myocardial injury. However, it remains to be fully elucidated how p53 works in cardiomyocytes.

【Methods & Results】

Neonatal rat cardiomyocytes were infected with adenoviral vectors expressing p53 and cultured for 48hr. DNA microarray analysis revealed that p53 overexpression resulted in the increased expression of inflammation-related genes, including CXCL10, an anti-angiogenic factor. RT-qPCR experiments showed that the expression of CXCL10 was upregulated by p53, while that of VEGF was not influenced. Next, we examined whether CXCL10 increased in HF animal models. In myocardial infarction (MI) model, the expression of CXCL10 was upregulated after MI both at acute and chronic stages, accompanied by the induction of p53. Moreover, in experimental autoimmune myocarditis models, CXCL10 expression was upregulated at chronic phase in myocardium specific STAT3 knockout mice which exhibited severe HF. Consistent with the previous studies, CXCL10 suppressed the migration of endothelial cells. Finally, since p53 is reported to exhibit anti-angiogenic property by interacting with HIF-1, we examined the effects of hypoxia on CXCL10 expression and found that hypoxia synergistically elevated the mRNA expression of CXCL10 with p53.

【Conclusion】

CXCL10, anti-angiogenic chemokine, is induced by p53, and this effect is enhanced by hypoxia pathways. Considering that impaired angiogenesis causes HF, CXCL10 could be a novel therapeutic target of HF.

Angiogenesis is essential for development and progression of tumors, and regarded as a rational target in anti-cancer treatment. We recently reported that L-type amino acid transporter 1 (LAT1) is upregulated in tumor-associated vascular endothelium of human pancreatic cancer and in vivo animal models. We also reported that tumor growth in animal model was significantly impaired through the inhibition of angiogenesis by targeting endothelial LAT1. To reveal the molecular mechanisms underlying the contribution of LAT1 in tumor angiogenesis, we investigated the effects of abrogating endothelial LAT1 on proliferation and intracellular signaling pathways in human umbilical vein endothelial cells. Pharmacological and genetic inhibition LAT1 drastically suppressed cell proliferation, causing a down-regulation of signaling pathways, GCAA- and mTORC1 pathways that are regulating the translation initiation. Furthermore, LAT1 was fundamental to promote migration, invasion, and tube formation induced by pro-angiogenic factor VEGF-A through the activation of mTORC1 in a manner independent to the tyrosine kinase receptor VEGFR2. These findings highlight a cross-talk between pro-angiogenic signaling and nutrient-sensing in tumor-associated endothelial cells, and may provide novel rational strategies for anti-angiogenic therapy.

G protein-coupled receptors (GPCRs) transduce their signaling through the activation of trimeric G proteins. The signaling is modulated by the interacting proteins with GPCRs, but the regulatory mechanisms are diverse and largely unknown. Parathyroid hormone receptor (PTHR) activates G_s, which in turn transduces adenylyl cyclase type 6 (AC6)/cyclic AMP (cAMP) pathway. We have previously shown that Tctex-1 (or DYNLT1) interacts with the carboxyl-terminus of PTHR. However, the role of Tctex-1 on PTHR/G_s signaling has remained unresolved. In the present study, we first found that knockdown of Tctex-1 did not alter the cell surface expression of PTHR without PTH-(1-34) stimuli in HEK293 cells stably expressing HA-tagged PTHR. In contrast, PTH-(1-34)-induced cAMP production was dramatically decreased by knockdown of Tctex-1. The Tctex-1-knockdown also suppressed cAMP production mediated through endogenous PTHR in MC3T3-E1 preosteoblast cells. Cyclic AMP production mediated through both PTHR and PTHR-KRVS mutant, which cannot bind to Tctex-1, was equally suppressed by Tctex-1-knockdown in HEK293 cells. The data indicate that the binding of Tctex-1 to PTHR is not a prerequisite for promoting cAMP production by Tctex-1. Moreover, cAMP production induced by forskolin, a direct AC activator, was reduced by Tctex-1-knockdown in HEK293 and MC3T3-E1 cells, suggesting that Tctex-1 augments AC activity. Finally, we found that Tctex-1 directly binds to AC6 in vitro. In summary, our results demonstrate that Tctex-1 augments PTHR/G_s/AC6 signaling through direct activation of AC6.

Post-translational S-palmitoylation by the zDHHC family of palmitoyl transferases impacts the function of thousands of proteins in animals and plants. We report here that nitric oxide (NO), which is generated in organisms across phylogeny, globally diminishes S-palmitoylation. Short-term exposure to NO S-nitrosylates zDHHCs and inhibits auto-palmitoylation of the catalytic Cys within the conserved DHHC active site. Longer-term NO exposure, in particular iNOS induction in activated macrophages promotes zDHHC down-regulation via lysosomal degradation. Thus, regulation by NO of the S-palmitolome may operate across phylogeny, and dysregulated S-palmitoylation likely represents a shared concomitant of the myriad human pathophysiologies characterized by enhanced production of NO.

Background: Tuberculosis (TB), caused by the bacillus Mycobacterium tuberculosis (Mtb), is the world’s leading infections disease. Because of increasing emergence of drug-resistant Mtb, development of a novel anti-TB drug is required. Bacterial proteases are maintaining proteastasis and essential for bacterial survival. Here, we investigated the feasibility of bacterial proteases as drug targets. 

Methods and Results:  Inducible knockdown strains of proteases (clp, ftsH or htrA) in Mycobacterium smegmatis mc²-155 were created by using CRISPR interference. Bacterial growth was not suppressed by gene knockdown, but knockdown of clp increased the susceptibility against isoniazid and rifampicin, traditional anti-TB drugs. Furthermore, checkerboad assay revealed the synergistic effect between cyclomarin A, a novel clp inhibitor, and anti-TB drugs.

Conclusion: Clp is a promising drug target for development of a novel anti-TB drug.

Clioquinol, a chelator and ionophore of copper/zinc, was extensively used as an amebicide to treat indigestion and diarrhea in the mid-1900s. However, it was withdrawn from the market in Japan because its use was epidemiologically linked to an increase in the incidence of subacute myelo-optic neuropathy (SMON). Yet, the pathogenesis of SMON has not been fully elucidated. A reporter assay revealed that clioquinol activated metal response element-dependent transcription in human neuroblastoma SH-SY5Y cells. Clioquinol significantly increased the cellular level of zinc within 1 h, possibly due to its ionophore effects. On the other hand, clioquinol significantly increased the cellular level of copper within 24 h. Clioquinol induced the oxidation of the copper chaperone antioxidant 1 (ATOX1), suggesting its inactivation and inhibition of copper transport. The secretion of dopamine-β-hydroxylase (DBH) and lysyl oxidase, both of which are copper-dependent enzymes, was inhibited by clioquinol, along with decreased noradrenaline levels. In fact, disruption of the ATOX1 gene suppressed the secretion of DBH. Taken together, the disturbance of cellular copper transport by the inactivation of ATOX1 may be one of the mechanisms involved in clioquinol-induced neurotoxicity in SMON.

Not Published

Sorghum bicolor (L.) Moench is known as a healthful food. We examined whether a water-soluble sorghum extract (SE) from S. bicolor has an anti-diabetic effect through a mechanism that improves insulin sensitivity or anti-adipogenesis. Although the treatment of SE did not affect the adipogenesis of 3T3-L1 adipocytes induced by isobutyl methylxanthine/dexamethasone/insulin (MDI), it significantly enhanced MDI/thiazolidinedione (TZD)-induced adipogenesis in 3T3-L1 adipocyte differentiation. Real-time PCR analysis showed that treatment with SE reduced the expression of adiponectin, adipocyte fatty acid-binding protein 2, and resistin in 3T3-L1 adipocyte cells. SE suppressed the expression of transcription factors, peroxisome proliferator-activated receptor γ (PPARγ), and CCAAT enhancer-binding protein α (C/EBPα）in both MDI- and MDI/TZDs-treated 3T3-L1 adipocytes. SE treatment reduced tumor necrosis factor α protein in cell lysates from MDI-induced 3T3-L1 adipocytes but not those induced by MDI/TZD. Our results suggest that SE can serve as an effective food source that improves insulin sensitivity and an anti-obesity agent.

Golgi fragmentation and ER exit site disassembly are considered to be the leading causes of the mitotic block of secretion from the ER. Although the mechanisms of Golgi fragmentation have been extensively characterized, ER exit block early in mitosis is not well understood. We previously reported that TANGO1 organizes ER exit sites by directly interacting with Sec16. We identified TANGO1 as a regulator of ER exit site disassembly during mitosis. TANGO1 phosphorylation was coordinately regulated by a kinase (CK1) and a phosphatase (PP1). CK1-mediated TANGO1 phosphorylation reduces binding to Sec16, leading to the disassembly of ER exit sites. CK1 constantly phosphorylates TANGO1, whereas PP1-mediated dephosphorylation of TANGO1 decreases during mitosis. Thus, the phosphorylation status of TANGO1, which is controlled by balanced activities of the kinase CK1 and the phosphatase PP1, regulates the organization of ER exit sites during the cell cycle.

Nerve growth factor (NGF) is one of the best-known examples of target-derived factors that regulate neuronal development. The biological processes of endocytosis and retrograde trafficking of NGF and its receptor, TrkA, are key mechanisms by which target-derived NGF influences neurons. We previously found the axon-derived TrkA elicits anterograde transport of naïve somatic TrkA via transcytosis. However, it is unclear whether the transcytosis machinery is a general mechanism which NGF utilizes to recruit additional membrane proteins necessary for axon growth and maturation. Here we report that amyloid-beta precursor protein (APP) is co-transcytosed with TrkA. Neurotrophin stimulation on distal axons induces co-localization and subsequent anterograde transport of somatic Trk receptors and APP. APP interacts with TrkA through its juxta-membrane domain, the domain that induces the formation of neurite-like processes in non-neuronal cells. The TrkA-APP interaction may also regulate proteolytic processing of APP. These data suggest that the TrkA-APP co-transcytosis is involved in NGF-dependent axon outgrowth and APP metabolism. Since APP and its proteolytic products play an important role in pathogenesis of Alzheimer’s disease (AD), our data further implicate that the deficit of TrkA-APP transcytosis is related to the onset of AD.

We previously reported that RNF183, a member of the RING finger (RNF) ubiquitin ligase family, is specifically expressed in the renal collecting duct and is induced under hypertonic conditions. However, its functional role under such conditions remains unclear. In this study, we used the BirA proximity-biotinylation technique to identify candidate substrates of RNF183; these included the basolateral Na-K-Cl cotransporter (NKCC1). We confirmed that RNF183 interacted with NKCC1 and ubiquitinated NKCC1 through K63-linked ubiquitination, which regulates protein transport. Bafilomycin A1, which inhibits lysosomal function, treatment of RNF183-expressing cells induced ubiquitinated NKCC1 accumulation, suggesting that NKCC1 are degraded in lysosomes by RNF183-mediated ubiquitination. In addition, RNF183 promoted the translocation of NKCC1 from the plasma membrane to intracellular region, compared to treatment with mock control or an RNF183 mutant. Furthermore, this translocation was facilitated under hypertonic conditions. Thus, RNF183 may play an important role in the adaptation to continuous hypertonic conditions through the downregulation of osmoregulatory transporters.

Previous investigations revealed that some probiotic bacteria exert anti-tumor effects in colorectal cancer. However, the anti-tumor molecules produced by probiotic bacteria have been less identified. In this study, we showed the strong anti-tumor effect of culture supernatant of Lactobacillus spices, and therefore identified ferrichrome as a tumor-suppressing molecule from the culture supernatant of L. casei using the HPLC separation system and mass spectrometry analysis. Anti-tumor effect of ferrichrome was superior than classical anti-tumor agents, including cisplatin and 5-FU. Ferrichrome induced the apoptotic reactions, including DNA fragmentation and cleavage of caspase-3 and PARP, in colorectal cancer cells. Transcriptome analysis revealed that ferrichrome induced DDIT3-mediated endoplasmic stress pathway. Ferrichrome exhibited an antitumor effect in a xenograft model, an AOM-DSS-induced carcinogenesis model and patient-derived organoid model. The biochemical test values were not markedly different when ferrichrome was administrated to mice. Therefore, we demonstrated that ferrichrome derived from L. casei suppressed the progression of colorectal cancer cell with little adverse effect, suggesting that it may be a novel agent for treating colorectal cancer.

Background and hypothesis: We recently found that knockout of SIRT1, a histone deacetylase, aggravates doxorubicin (DOX)-induced cardiotoxicity. We hypothesized that SIRT1 regulates DNA damage response (DDR) to attenuate cardiotoxicity.

Methods and Results: Wild type (WT) and SIRT1 knockout in the cardiomyocyte (SIRT1-cKO) mice were treated with vehicle or DOX (4 IP injections of 5 mg/kg/week) starting at 3-month-old.  Immunoblotting showed that DOX increased myocardial levels of phospho-Ser139-histone H2AX (pS139-H2AX), a critical mediator of DDR, in WT. However, the increase in pS139-H2AX by DOX was attenuated in SIRT1-cKO. The nucleus with DNA fragmentation evaluated by TUNEL staining was more increased by DOX in SIRT1-cKO than WT (0.384 vs 0.194%).  In H9c2 cardiomyocytes, cell death after DOX was enhanced by knockdown (KD) of SIRT1. DOX increased pS139H2AX levels, which was attenuated by SIRT1-KD. Pharmacological inhibition or KD of SIRT1 increased acetyl-Lys5 (K5) H2AX level. Furthermore, S139-H2AX phosphorylation by DOX was attenuated in a mutant H2AX with acetylation mimic of K5 (K5Q-H2AX) compared with that in WT-H2AX.

Conclusion: These suggest that deacetylation of H2AX at K5 by SIRT1 plays a critical role in Ser139-H2AX phosphorylation to promotes proper DDR and therefore to attenuate cardiotoxicity.

Objective Nitric oxide (NO) is an essential neurotransmitter in the lower urinary tract (LUT). Since it is known that NO deficiency leads to LUT dysfunction (LUTD), supplementary of NO might be an effective therapy for LUTD. However, there are some issues in the biological application of NO. Firstly, it is difficult to control the timing of NO action, because of its short half-life and rapid diffusion. Secondly, NO leads to systemic side effects such as headache or hypotension. To resolve these issues, we developed a red-light reactive NO donor, “NORD-1,” with which we could regulate the release of NO temporally and spatially. In this study, we aimed to examine the possibility to apply NORD-1 to LUTD.

Methods We used 10-11-week-old Wistar/ST rats. We injected NORD-1 or vehicle into the rats’ bladder and used those rats for the experiments after twenty minutes. As in vitro study, we performed the isometric tension study on the bladder neck and bladder body specimens. Also, we performed cystometry to evaluate the function of the bladder as in vivo study.

Results The specimens of the vehicle group did not react to the light irradiation. The bladder neck specimen of the NORD-1 group was relaxed during light irradiation. In contrast, the bladder body specimen of the NORD-1 group did not react to the light irradiation. On the cystometry, the micturition intervals were prolonged during the irradiation.

Conclusion We succeeded to control both the bladder neck relaxation and the micturition of rats using NORD-1 and red-light irradiation. NORD-1 may be a novel therapeutic agent for LUTD.

[Aim] High salt intake is a risk factor for urinary storage symptoms. In this study, we investigated the effects of high salt intake on detrusor muscle contraction in rats.

[Methods] Six-week-old male Dahl salt-resistant (DR, n = 5) and Dahl salt-sensitive (DS, n = 5) rats were fed a high salt diet (HS; 8% NaCl containing CE-2) for 1 week. The contractile responses of the detrusor muscle to cumulative administration of carbachol and electrical field stimulation (EFS) in the absence and presence of inhibitors (suramin; a purinergic receptor inhibitor, atropine; a muscarinic receptor inhibitor) were evaluated by measuring isometric tension.

[Results] Carbachol concentration versus response curves shifted to lower concentrations in the DS + HS group than those in the DR + HS group. Contractile responses to EFS were more enhanced in the DS + HS group than those in the DR + HS group. There was no difference in the purinergic components-induced contractile responses, whereas cholinergic components-induced contractile responses were more enhanced in the DS + HS than those in the DR + HS group.

[Conclusion] One week of high salt intake in DS rats enhanced detrusor muscle contraction via an increased sensitivity to cholinergic signals. Enhanced detrusor muscle contraction might lead to urinary storage symptoms.

Diabetic nephropathy (DN) is a leading cause of end-stage renal disease (ESRD). Hypertension increases kidney stress, which deteriorates function, and leads to peripheral renal vascular resistance. Long-term hypoperfusion promotes interstitial fibrosis and glomerular sclerosis, resulting in nephrosclerosis. Although hypertension and DN are frequent ESRD complications, relevant animal models remain unavailable. We generated a deoxycorticosterone acetate (DOCA)-salt hypertensive uni-nephrectomized (UNx) KKA^y mouse model demonstrating hypertension, hyperglycemia, cardiac hypertrophy, kidney failure, increased urinary albumin creatinine ratio (UACR), and increased renal PDE4D and cardiac PDE5A mRNA levels. We hypothesized that the novel PDE4 selective inhibitor, compound A, and PDE5 inhibitor, sildenafil, exhibit nephroprotective and cardioprotective effects in this new model. Compound A, sildenafil, and the angiotensin II receptor blocker, irbesartan, significantly reduced ventricular hypertrophy and pleural effusion volume. Meanwhile, compound A and sildenafil significantly suppressed the UACR, urinary kidney injury molecule-1, and monocyte chemoattractant protein-1 levels, as well as that of renal pro-fibrotic marker mRNAs, including collagen 1A1, fibronectin, and transforming growth factor-beta (TGF-β). Moreover, compound A significantly suppressed TGF-β-induced pro-fibrotic mRNA expression in vitro in all major kidney lesions, including within the glomerular mesangial region, podocytes, and epithelial region. Hence, PDE4 and PDE5 inhibitors may be promising treatments, in combination with irbesartan, for DN with hypertension as they demonstrate complementary mechanisms.