The hippocampus that includes neural stem cells (NSCs) is an important organ associated with memory and learning. As a simple retrospective assay to identify NSC activity, neurosphere culture is generally employed. However, it is difficult to expand stemness-possessing neurosphere from the adult hippocampus. To better understand the nature of adult hippocampal NSCs, establishment of the specified in vitro expansion system is needed.

Here, we tried the culture of NSCs on a polyimide sheet possessing pores ranging from 5 to 50 µm. The NSCs derived from the adult hippocampus efficiently proliferate and sparsely formed sphere-like aggregates on the sheet. Those aggregates detached from the sheet could differentiate into the three cell lineages in response to differentiating agents. According to this procedure, we enabled the adult hippocampus-derived NSCs to maintain their stemness at least over 400 days. To evaluate the molecular mechanism, we performed a DNA microarray analysis. Notably, there was a big difference in expression profiles of mRNAs from NSCs between the long-term 3D culture and the short-term spheroid culture. We will discuss how the stemness of NSC can be maintained by the culture with polyimide sheet.

The development of neuroprotective agents is necessary for the treatment of neurodegenerative diseases. Here, we report PQA-11, a prenylated quinolinecarboxylic acid (PQA) derivative, as a potent neuroprotectant. PQA-11 inhibits glutamate-induced cell death and caspase-3 activation in hippocampal cultures, as well as inhibits N-Methyl-4-phenylpyridinium iodide- and amyloid β_1-42-induced cell death in SH-SY5Y cells. PQA-11 also suppresses mitogen-activated protein kinase kinase 4 (MKK4) and c-jun N-terminal kinase (JNK) signaling activated by these neurotoxins. Quartz crystal microbalance analysis and in vitro kinase assay reveal that PQA-11 interacts with MKK4, and inhibits its sphingosine-induced activation. The administration of PQA-11 by intraperitoneal injection alleviates 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced degeneration of nigrostriatal dopaminergic neurons in mice. These results suggest that PQA-11 is a unique MKK4 inhibitor with potent neuroprotective effects in vitro and in vivo. PQA-11 may be a valuable lead for the development of novel neuroprotectants.

Diabetic retinopathy results in visual dysfunction through retinal ganglion cell (RGC) death. We have previously shown that APJ, an apelin receptor, is expressed in the RGCs and intravitreal injection of apelin protects against NMDA-induced RGC death. In the present study, we investigated whether systemic administration of an APJ agonist prevents RGC death in the retina of the diabetes model mouse fed the high fat diet (HFD).  We used Insulin2 mutant (Ins2+/-) Akita mouse, which is a mouse model of type 1 diabetes. The Akita mouse was fed the HFD from 5 weeks after birth. The APJ agonist ML233 (5 mg/kg) was administered intraperitoneally on every other day for 4 weeks from 5 weeks after birth. The electro-responses of the RGCs were measured by electroretinography. ML233 suppressed the reduction of both the electro-response and the number of Brn-3a positive RGCs in the retina of the Akita mice fed HFD for 4 weeks. These effects were blocked by an APJ antagonist ML221 (5 mg/kg, i.p.). The present study showed that the APJ agonist protected against RGC death via APJ in the diabetes model mouse fed the HFD, suggesting that systemic administration of APJ agonists may prevent RGC degeneration in diabetic retinopathy.

Nanomaterials have a variety of unique physical and chemical properties, and are being studied for biotechnological, pharmacological, and medical applications. Silica nanoparticles (SiNPs) are produced on an industrial scale and used in various fields. Despite these benefits, there is concern that exposure to SiNPs may lead to adverse effects in certain types of cells or tissues, such as developmental abnormalities in the brain and developing embryos. Although investigations on the toxicity of SiNPs against neurons are essential for medicinal use, a few studies have assessed the direct effects of SiNPs on cells derived from the central nervous system. In this study, we investigated the toxic effects of SiNPs on primary cultures of hippocampal cells. We showed that treatment with SiNPs caused oxidative stress and cell death. Furthermore, we found that these cytotoxicities were dependent on the particle size, concentration, and surface charge of SiNPs. The toxicity was reduced by SiNP surface functionalization or protein coating and by pretreating cells with an antioxidant, suggesting that contact-induced oxidative stress may be responsible for SiNP-induced cell death. These data will be valuable for utilizing SiNPs in biomedical applications.

Gastrin-releasing peptide (GRP) receptor (GRPR)⁺ neurons in the spinal dorsal horn (SDH) are itch-responsive, but their regulatory mechanisms are not yet clarified. Recently, we found that not only GRP but also glutamate directly activate GRPR⁺ neurons through AMPA receptors (AMPAR), and those transmitters play fundamental roles in the spinal transmission of physiological itch. Here, we determined whether GRP-GRPR system underlies pathological itch in mice. To induce contact dermatitis, diphenylcyclopropenone (DCP) was applied to C57BL mice after having shaved on the back. The mRNA expression levels of GRP and GRPR in the cervical SDH were upregulated after DCP application. DCP-induced scratching behaviors were prevented by ablation of GRPR⁺ AMPAR⁺ neurons following bombesin-saporin treatment. Moreover, chemogenetic silencing of GRP⁺/glutamate⁺ neurons using Cre-dependent Gi- designer receptors exclusively activated by designer drugs (DREADD) also attenuated DCP-induced scratching behaviors. These results suggest that GRP-GRPR system and glutamate-AMPAR system in the SDH might cooperatively regulate not only physiological but also pathological itch.

Glycosphingolipids that have diverse variations in their carbohydrate-chains are abundant in neural tissues and previous studies revealed the important roles in neuronal functions. However, the roles of glycosphingolipids in the pain sensing pathway remain unclear. We reported that sialic acid-containing glycosphingolipids, gangliosides, are involved in nociceptive behavior. Depending on carbohydrate structures of gangliosides, they are divided into four groups such as o-, a-, b-, and c-series. Intraplantar injection of GT1b (b-series gangliosides) but not GM1a (a-series gangliosides) caused mechanical allodynia that was attenuated by the TRPV1 antagonist capsazepine. In addition, pretreatment of GT1b enhanced capsaicin induced nociception. On the other hand, intradermal injection of GM1a enhanced chloroquine induced pruritus.

These results suggested that the differences in carbohydrate structures of gangliosides lead to different effects on cutaneous sensation.

The large-conductance Ca²⁺- and voltage-activated K⁺ (BK) channel is formed by four pore-forming subunits encoded by a single Slo1 gene and dually regulated by membrane voltage and intracellular Ca²⁺ levels. Recently, accumulating evidence using pharmacological methods suggests that the BK channel are associated with nociceptive sensitization. However, mechanisms underlying the effect of the BK channel on the regulation of pain signaling is still largely unknown. Here, we studied the role of the BK channel in the pain signaling in vivo using mice deficient for Slo1. In a partial sciatic nerve ligation (PSNL) model, Slo1 KO mice showed partially restored thermal hyperalgesia compared to their WT littermates. In microglia from WT mice, p38 MAPK phosphorylation was significantly increased after lysophosphatidic acid (LPA) treatment; conversely, it was not significantly upregulated in microglia from Slo1 KO mice, suggesting that the BK channel contributes to the signaling of microglial hyperactivation. Notably, intrathecal (i.t.) injection of microglia derived from WT mice into Slo1 KO mice before PSNL induced the normal development of hyperalgesia in Slo1 KO mice. These results indicate that the BK channel activation in spinal microglia, but not in neuron, contribute to the induction of neuropathic pain in this PSNL model.

Oxaliplatin, a platinum-based chemotherapy drug, often induces cumulative sensory peripheral neuropathy. Accumulating evidences suggest a possible relationship between vascular impairment and peripheral neuropathy. In this study, we examined the effects of vasodilators on the oxaliplatin-induced peripheral neuropathy in mice. Mice injected with oxaliplatin (10 mg/kg, i.p.) once a week for 8 weeks showed mechanical (von Frey filament test) and cold (acetone test) hypersensitivities. A single injection of vasodilators (PDE5 inhibitor, endothelin receptor antagonist and prostaglandin analog) 1 h before tests improved the decreased skin blood flow and cold hypersensitivity induced by oxaliplatin, while it had no effect on mechanical hypersensitivity. When mice were fed with diets containing PDE5 inhibitor for 8 weeks during oxaliplatin treatment, the vascular impairment and hypersensitivities to mechanical, cold and transcutaneous current stimuli were suppressed. Furthermore, it prevented the decrease in nerve conduction velocity and axonal degeneration induced by oxaliplatin. These results suggest improvement by PDE5 inhibitor in vascular impairment can prevent oxaliplatin-induced peripheral neuropathy.

Schwann cells (SC) generate myelin sheath in the peripheral nervous system. SC differentiation stages observed during development as well as degeneration/regeneration of the peripheral nerve have been characterized by the expression of genes specific for each stage. However, it remains unclear how these genes regulate SC differentiation and peripheral myelination. Here, we show the involvement of hypoxia induced factor-1α (HIF-1α) in the regulation of SC differentiation and myelination. Expression of HIF-1α in SC increased during development of peripheral nerves. Hypoxic treatment, application of a HIF-1α stabilizing drug, and overexpression of HIF-1α, bearing a mutation which gives resistance to proteasomal degradation, activated the expression of myelin related genes in SC and facilitated myelination in vitro. Expression of HIF-1α was also observed in SC in peripheral nerves after injury. In addition, the number of myelinating axons was increased by an local application of a HIF-1α stabilizing drug. These findings suggest that HIF-1αmight be involved in SC differentiation and peripheral myelination during development as well as regeneration after injury. We are now investigating whether HIF might be a potential therapeutic target for inherited neuropathy such as Charcot-Marie-Tooth disease.

Uric acid (UA) is well known as the end product of purine metabolism in human. Hyperuricemia is defined as a serum UA level of ≥ 7.0 mg/dl (416 µM) in Japanese guideline for the management of hyperuricemia and gout (second edition), which is known as a risk factor for cardiovascular disease. Recently, it has been reported that low UA levels as well as high UA levels are predictive markers of increased mortality in epidemiologic studies. Hence, the aim of this study is to assess the role of UA at physiological concentration (conc) in normal cells. Normal human dermal fibroblasts were treated with UA conc at 62.5 - 500 µM (1.1 - 8.4 mg/dl). Cell viability of UA treated groups within the normal range was higher than that of control group. There was no change in cell number among groups, but total protein levels in UA treated groups increased at 72 hours. On the other hand, UA is known to have the potential antioxidant effects. Physiological conc of UA treatment decreased reactive oxygen species in the present study. Additionally, the expression of an oxidative stress-related protein was increased by UA treatment. Taken together, these findings suggest that physiological conc of UA in normal cells is probably implicated in cell viability.

The differences of purine metabolism of mice from that of human are known to be not only conserved uricase (Uox), but also low hypoxanthine phosphoribosyltransferase (HPRT) activity. The aim of this study is the establishment of high HPRT activity-Uox knockout mice as mouse model for the study of purine metabolism in human and the investigation of the effect of Allopurinol and Topiroxostat on HPRT. Allopurinol 30 mg/kg (Allo) and Topiroxostat 1 mg/kg (Top) were administered to the model mice for 7 days by feeding diet. Oxypurines (urate (UA), hypoxanthine (HX) and xanthine (XA)) and creatinine (Cr) in plasma and urine were measured by HPLC. Plasma UA value and urinary UA/Cr ratio significantly decreased in Allo and Top. Although the plasma UA-lowering effect was equivalent in Allo and Top, the urinary HX+XA/Cr ratio in Top 1 was significantly lower than those in Allo. Moreover, urinary oxypurine/Cr ratio demonstrated a significant lowering effect in Top, but not in Allo. In conclusion, Topiroxostat has a potent plasma UA-lowering effect and didn't affect the salvage pathway unlike Allopurinol, efficiently resulted in decreased total oxypurine excretion.

Although xanthinuria is asymptomatic in humans, xanthine dehydrogenase knockout (XdhKO) mice die from renal failure. The activities of hypoxanthine phosphoribosyl transferase (HPRT) of human and wild mice are higher than those of labolatory mice. The aim of this study is to compare the life span between the XdhKO mice with high and low HPRT activity. High HPRT activity XdhKO mice were produced by crossbreeding with a consomic C57BL/6 mice whose Hprt allele is derived from wild mice. Urinary substances of high or low HPRT activity XdhKO mice were separated by HPLC. In a low HPRT mice, which died at 7 weeks of age, the excretion of xanthine (XA) decreased with increasing excretion of hypoxanthine (HX) without changing the total amount of urinary oxypurine excretion. At one week before death, HX excretion of low HPRT mice were 2.5 times higher than those of high HPRT mice. Prior to death of high HPRT activity mice, the excretion of XA and its precursor xanthosine decreased as HX excretion increased. In conclusion, the change of urinary oxypurine excretion from xanthine to hypoxanthine might be a cause of death of XdhKO mice, suggesting involvement of the reduction of IMP dehydrogenase activity due to NAD⁺ deficiency or competitive inhibition.

Advanced glycation end-products (AGEs), which comprise non-enzymatically glycosylated proteins, play an important role in several diseases and aging processes including angiopathy, renal failure, diabetic complications, and neurodegenerative diseases. Among AGE-associated phenotypes, toxic AGEs, glyceraldehyde-derived AGE-2, and glycolaldehyde-derived AGE-3 are involved in the pathogenesis of diabetic complications. In addition, macrophages are reported to remove extracellular AGEs from tissues via scavenger receptors, leading to the progression of atherosclerosis. In the present study, we found that AGE-2 and AGE-3 enhanced their own endocytic uptake by RAW264.7 mouse macrophage-like cells in a concentration-dependent manner. Furthermore, we demonstrated the morphology of phagocytic macrophages and the endocytosis of AGE particles. The toxic AGEs induced the expression of a scavenger receptor, CD204/scavenger receptors-1 class A (SR-A). Notably, an antibody against CD204 significantly prevented toxic AGE uptake. Moreover, an SR-A antagonistic ligand, fucoidan, also attenuated the AGE-2- and AGE-3-evoked uptake in a concentration-dependent manner. These results indicated that SR-A stimulation, at least in part, plays a role in AGE uptake.

【Background】

Recently, even dogs have developed an aging society. It is recognized that oxidative stress is relative to aging and disease. Many traditional Chinese medicines have been said that they had anti-oxidantive effect. But, there are no repot that these medicines have anti-oxidative effect in dogs. In this study, therefore, I examined anti-oxidative effect of Juzen-taiho-to, one of the traditional Chinese medicines, in dogs.

【Material and Method】

Five healthy female beagle dogs (38-41months of age and weighing 8.6-10.7 kg) were orally administered Juzen-taiho-to at 450 mg/kg with food for 28 days. Blood samples were taken from five dogs at day0, 7, 14, 21 and 28. Using the blood samples, anti-oxidant level, oxidative stress level and blood fluidity were determined.

【Results / Discussion】

Although improvement tendency was seen in anti-oxidant level and blood fluidity, the significant difference was not observed. But oxidative stress level at day 14, 21, and 28 was significantly lower than day 0. So, Juzen-taiho-to may have anti-oxidative effect in dogs by reducing oxidative stress. In addition, because traditional Chinese medicine is habitually administered for long term, further effects may be expected by administering Juzen-taiho-to in dogs for longer term.

Characterization of mononuclear cardiomyocytes in adult murine hearts

【Background and Objective】

Mammalian adult hearts have poor regenerative ability and the injured hearts scarcely recover. However, in adult mice, mononuclear cardiomyocytes (mnCMs), which account for about 10% of total cardiomyocytes, are relatively more proliferative than polynuclear cardiomyocytes (pnCMs). Therefore, we focused on mnCMs and examined their properties.

【Methods and Results】

We tried to analyze genes that are highly expressed in mnCM group. Cardiomyocytes from adult mice were separated into mnCM and pnCM groups by single cell handling device, followed by RNA-sequence analysis. As a result, about 480 genes, whose expression was markedly increased by more than 5 folds in the mnCM group as compared with the pnCM group, were extracted. These extracted genes included 4 marker genes characteristic of immature cardiomyocytes, such as Myl7 and Nppa. Next, focusing on Myl7, fluorescent immunostaining was performed, and the expression of Myl7 was observed in small myocyte population.

【Discussion】

It could be concluded that mnCMs population includes subpopulation that expresses marker genes characteristic of immature cardiomyocytes, such as Myl7. Further studies would be required to elucidate the biological function of these immature cardiomyocytes.

The overexpression of CGRRF1 induces hypertrophy in cultured cardiomyocytes

【Background】Cardiomyocytes (CMs) are terminally differentiated cells that lose proliferative capacity immediately after birth; however, it remains to be fully elucidated how CMs exit from cell cycle and reach maturity. In this study, among the factors involved in cell cycle arrest, we focused on Cell Growth Regulator With Ring Finger Domain 1 (CGRRF 1).

【Objective】To explore the biological significance of CGRRF1 in CMs.

【Methods and Results】Real time RT-PCR analyses demonstrated that the expression of CGRRF1 mRNA was increased in murine adult hearts, compared with neonatal hearts.  Next, we examined the expression of CGRRF1 mRNA in adult and neonatal CMs and found that CGRRF1 expression was remarkably upregulated in adult CMs. Since the expression level of CGRRF1 increases in cells expressing p53, neonatal rat CMs were infected with adenoviral vector expressing p53. The overexpression of p53 enhanced expression of CGRRF1 mRNA. Finally, in order to investigate the biological function of CGRRF1, we constructed adenoviral vector expressing CGRRF1. Interestingly, CGRRF1 overexpression resulted in CM hypertrophy.

【Discussion】CGRRF1 was induced in CMs during growth. The overexpression of CGRRF1 enlarged CMs, suggesting that CGRRF1 may be related to CM maturation.

Introduction

Murine ventricular cardiomyocytes (CM) are still immature at birth. Within 1 month after the birth, they are maturated through physiological hypertrophy, enrichment of sarcoplasmic reticulum and formation of t-tubules bearing abundant L-type Ca²⁺ channels. This channel is involved in Ca²⁺-induced Ca²⁺ release that causes maturated excitation-contraction and forceful ventricular contraction. In heart, the concentration of several cytokines, growth factors and hormones are known to change after birth. However, it is unclear which of the factors are responsible for the final CM maturation in mice.

Methods

The effects of various inhibitors of receptors for these factors on final CM maturation were assessed in P1-20 mice. Their cardiac function was evaluated with echocardiogram. Morphology of t-tubules and a twitch Ca²⁺ transient were analyzed in the isolated CM.

Result

Cardiac function was significantly lower in mice treated with nintedanib, an inhibitor of FGF, PDGF and VEGF receptors, or SC-144, gp130 antagonist than control mice. The nintedanib-treated mice also exhibited smaller peak of Ca²⁺ transients. We are intending to determine which of the factors are necessary for the CM maturation by using shRNA delivered with adeno-associated virus in living mice.

The early stage of diabetes mellitus (DM)-related cardiomyopathy (DMCM) is characterized by left ventricular (LV) diastolic dysfunction. The aim of this study was to elucidate the mechanism of the defective Ca²⁺ signaling underlying diastolic dysfunction in DMCM. In the streptozotocin (STZ)-induced DMCM model mice 4 weeks after STZ treatment, diastolic function was impaired without reduction of ejection fraction. In the isolated LV myocytes from DM mice, the Ca²⁺ transient decay rate was slower than that from control. In the ventricle of DM mice, the expression level of junctophilin2 protein was significantly lower, although expression levels of Ca_V1.2, RyR2 and SERCA2 were the same as those of control mice, suggesting that uncoupling of dyad junction starts at the early stage. The phosphorylation level of phospholamban (p-PLN) was significantly lower. Insulin treatment recovered the p-PLN level and the relaxation rate of the isolated ventricular myocardium from DM mice. Furthermore, PKA-independent insulin/PKG signaling turned out to be required for maintaining basal p-PLN. These results indicate that the reduction of p-PLN caused by insulin signaling defect is responsible for the LV diastolic dysfunction in the early stage of DMCM.

Recent findings highlight the unexpected toxicity of anticancer drugs on left ventricular (LV) function. Nevertheless, a strategy to predict the potential toxicity of drugs on LV function is still limited. In the present study, we developed a novel culture system with patterned human induced pluripotent stem cell-derived cardiomyocytes (hiPSCMs) in a 96 well format and evaluated the acute-to-chronic effects of several anticancer drugs by our system. We found that doxorubicin exhibited chronic, not acute, robust toxicity on the movement of hiPSCMs. The toxicity of molecule-targeted anticancer drugs was generally mild, however, sunitinib impaired the contraction, relaxation and synchronicity of contraction more strongly than other molecule-targeted anticancer drugs. We also identified that synchronicity of contraction was impaired only by the anticancer drugs with warning on LV toxicity. Patterned hiPSCMs also exhibited distinct, mechanism of action-based, contractile responses against various cardioactive compounds. These results collectively suggest the usefulness of patterned hiPSCMs in assessing the effect of drugs on LV function.

Apolipoprotein E-containing lipoproteins (E-LPs) have crucial roles of lipid transport in the central nervous system. α2-Macroglobulin (a2M) is a pan-protease inhibitor found in plasma and cerebrospinal fluid. Both of these proteins act as ligands of low density lipoprotein receptor-related protein 1 (LRP1). We previously reported that E-LPs protected neurons via the LRP1 from degeneration induced by glutamate excitotoxicity in vitro and in vivo. It has been reported that a2M is increased in aqueous humor of glaucoma patients. Here, we demonstrated that intravitreal injection of E-LPs protected retina from degeneration caused by intravitreal injection of N-methyl-D-aspartate in rats, a model of glaucoma. Intravitreal administration of E-LPs also attenuated the elevated a2M level in aqueous humor of the glaucoma model. Moreover, an addition of E-LPs into medium of primary cultured retinal glia decreased a2M level through LRP1. The protective effect of E-LPs against excitotoxicity in primary cultured retinal ganglion cells was interfered by a2M, but additional treatment of E-LPs overcame the interference. These results may indicate a potential therapeutic role of an LRP1 ligand in the retina as treatments for glaucoma.

Amyloid β (Aβ) skews microglia to M1 phenotype and induces inflammation and neurodegeneration. On the other hand, another type of microglia, M2, shows anti-inflammatory and neurotrophic effects. We previously clarified that HDAC3 inhibition induced predominance of M2 microglia and axonal growth, and recovered locomotor function in spinal cord injured mice. Therefore, this study aimed to clarify that HDAC3 inhibition skewed to M2 microglia and restored memory function in Alzheimer's disease model mice. An HDAC3 inhibitor, RGFP966 was intraperitoneally administered to 5XFAD mice, a transgenic model of Alzheimer's disease. RGFP966 improved novel object recognition memory in 5XFAD mice. When microglia in the brain of 5XFAD mice were eliminated by intracerebroventricular administration of clophosome, the effect of RGFP966 was diminished. In cultured microglia, RGFP966 treatment skewed to M2 microglia when treated 24 h after Aβ addition. Conditioned medium was collected from RGFP966-treated microglia, which recovered Aβ-induced collapse of axonal growth cones. These results suggest that HDAC3 inhibition increased predominance of M2 microglia, recovered axonal degeneration, and ameliorated memory deficit in 5XFAD mice.

Enhanced production of pro-inflammatory cytokines by inflammasome activation in microglia has been shown to induce neuronal damage in Alzheimer's disease (AD). Impairment of neurogenesis has been also shown to contribute to cognitive decline in AD. Recent reports showed that inflammasome activation is induced in mouse neural progenitor cells (NPC). Thus, we determined involvement of inflammasome activation on proliferation or differentiation of NPC. Activation of inflammasomes in NPC was examined by the expression of inflammasome components (NLRP3, ASC, or caspase-1), IL-1beta, or IL-18. Proliferation of NPC was examined by MTT assay. The differentiation potential of NPC into neural cells was evaluated by NeuN expression using western blot analysis. Stimulation with TNFalpha and LPS induced inflammasome activation and production of IL-1beta and IL-18 in mouse NPC. Stimulation with TNFalpha and LPS significantly inhibited the proliferation of NPC and NeuN expression in the differentiated NPC. The treatment with either IL-1beta or IL-18 significantly inhibited NeuN expression in the cells. Thus, the inflammasome activation may inhibit proliferation and neural differentiation in mouse NPC via the enhanced production of IL-1beta or IL-18.

Neurotoxic soluble amyloid-beta 42 (Aβ) oligomers are increasingly believed to cause synaptic failure in the hippocampus, leading to memory disability, in the early stages of Alzheimer's disease (AD). Hippocampal somatostatin (SST)-neprilysin (NEP) system is known to function as a defense system against the neurotoxic Aβ oligomers, and to be compromised due to aging and AD pathogenesis. Therefore, restoration of this compromised hippocampal SST-NEP system may enable prevention of onset of AD and progression of the illness. We originally found that nobiletin-rich Citrus reticulata peel also known as Nchinpi did prevent animal memory defect. Consistently, our pilot clinical study suggested its beneficial potential in patients with AD, without adverse side effects, including digestive symptom. In the present study in the hippocampus of aged mice as well as in primary cultures of hippocampus neurons, we therefore evaluated the impacts of Nchinpi extract on expression of SST and NEP genes. Treatment with Nchinpi extract did coordinately facilitate SST and NEP levels in the cultured neurons, as assayed by real-time RT-qPCR and ICC; and it raised mRNA levels for both genes tested in the hippocampus, when the Kampo medicine was orally given to aged (17-month-old) mice at a dose of 0.5 g/kg/day for 14 consecutive days. IHC uncovered the ability to raise neuronal SST- and NEP-immunoreactivities. These findings thus suggest that the Nchinpi extract highly likely prevents such an age-related decline in the hippocampal SST-NEP system's function.

The spread of abnormal protein in neurodegenerative diseases has been shown to be strongly correlated with clinical phenotypes, and it has been pointed out that close relationship with disease progression. Recently, abnormal proteins caused by neurological diseases have the same properties as "prion" of prion diseases. Prion or prion-like proteins may convert normal molecules into abnormal forms, proliferate and propagate between cells.  Abnormal proteins such as tau or α-synuclein that accumulate in brains with dementia have been shown to propagate like prion proteins.  However, the expression patterns of tau in the mouse brain are different from those in humans, and the pathogenesis in the animal model of abnormal tau propagation remains incomplete.  To overcome this problem, a novel mouse showing tau expression patterns similar to those of humans was developed using genome editing techniques.  We inoculated the brain of this mouse with a sarkosyl-insoluble fraction containing abnormal tau derived from tauopathy patients and examined the accumulation of tau pathologies.  We also performed a detailed analysis of the relationship between the inoculation site and sites where tau accumulates abnormally by histochemical and neuronal circuitry, and elucidated the propagation mechanism of the abnormally accumulated protein.  This research is expected to lead to the development of novel drugs for dementia using the novel approach of "inhibition of abnormal protein propagation".

Organelles extensively communicate with each other to regulate cellular responses. The aim of the present study was to reveal the involvement of organelle communication in regulation of host defense responses. In particular, we focused on the functions and roles of organelle communication in regulation of innate immune response. The innate immune system senses pathogens and induces the release of inflammatory mediators leading to the induction of host defense responses. However, the innate immune system often induces an aberrant inflammatory response, which causes severe tissue damage. Therefore, understanding the mechanisms underlying the regulation of innate immune system is required for identifying promising targets for developing anti-inflammatory drugs. We observed that communication among organelles such as lysosomes and mitochondria was involved in the regulation of innate immune response. We also observed that compounds targeting organelle communication-mediated innate immune response blocked inflammation-induced tissue damage. Here, we will discuss the spatial regulation of innate immune response and anti-inflammatory effects of compounds targeting it from the perspective of organelle communication.

Advanced glycation end-products (AGEs), especially toxic AGEs derived from glyceraldehyde (AGE-2) and glycolaldehyde (AGE-3), are biologically reactive compounds. Accumulated evidence suggests that the AGEs are associated with both microvascular and macrovascular complications in diabetic mellitus. Macrophages are reported to remove extracellular AGEs from tissues via scavenger receptors, leading to the progression of atherosclerosis. In the present study, we found that AGE-2 and AGE-3 enhanced their own endocytic uptake by RAW264.7 mouse macrophage-like cells. An antibody against scavenger receptors-1 class A (SR-A, CD204) significantly prevented toxic AGEs uptake. In contrast, none of neutralizing antibody against LOX-1, CD163, CD206, RAGE and CD36 has inhibitory activity on uptake of toxic AGEs. In addition, we showed that algae-derived fucoidan or carrageenan and artificially-produced dextran sulfate which is known as an SR-A antagonistic ligand, but not the others sulfated polysaccharide, including heparin, chondroitin sulfate and hyaluronic acid, has inhibitory activities on uptake of toxic AGEs. These findings suggest that the SR-A is partially involved in the toxic AGEs uptake.

Kikyo-to is a kampo medicine which is composed of Platycodi Radix (PR) and Glycyrrhizae Radix (GR), and used for Pharyngitis. As previous research in our laboratory has found the possibility that kikyo-to could influence cell viability of RAW264.7, we examined the effect of kikyo-to in detail.

Decoction of both crude PR and GR, that is kikyo-to (K-D), or each crude herbal medicine, or aqueous solution of extract granule preparation of kikyo-to (K-G) were exposed to RAW264.7 mouse macrophage-derived cells, CACO-2 human colorectal cancer cells and K562 human blood cancer cells. Viability of these cells were evaluated after exposure of various dilution of these preparations.

K-D decreased cell viability of RAW264.7 in a time and concentration-dependent manner and K-G also decreased cell viability of RAW264.7 in a concentration-dependent manner. In contrast, the viability of other cells did not change significantly. Decoction of GR alone decreased cell viability of RAW264.7, suggesting that components of GR may contribute to decrease in cell viability of RAW264.7, selectively. To confirm this hypothesis, further studies, functional analysis and clinical validations are needed.

The plasma levels of inter-α inhibitor proteins (IAIPs) are decreased in patients with sepsis and the reduced levels correlate with increased mortality. In the present study, we examined the beneficial effects of IAIPs on human neutrophils in the treatment of sepsis. We demonstrated that IAIPs induced a spherical shape of neutrophils that was smaller in size with a smooth cellular surface in a concentration-dependent manner. IAIPs inhibited the spontaneous release of reactive oxygen species (ROS) in a concentration-dependent fashion. ROS inhibition was associated with reduction in p47phox phosphorylation on Ser328. IAIPs inhibited the NETosis that is alive consistent with the concentration-dependent inhibitory effects of IAIPs on ROS production. The inhibitory regulation of CD162 (PSGL-1) expression by IAIPs demonstrated that the suppressive effects of IAIPs on the interaction between neutrophils and other effector cells attenuates the inflammatory response. IAIPs also inhibited Zn²⁺-induced PS expression and aggregation of erythrocytes. Our results suggest that IAIP-induced morphological changes that render neutrophils quiescent, reduce production of ROS, inhibit immunothrombosis during polymicrobial infections and erythrocytes aggregation. Thus, IAIP plays a key role in controlling neutrophil activation and eryptosis.

The Ca²⁺-activated K⁺ channel (K_Ca3.1) is one of key molecules that control Ca²⁺ entry, and the increase of Ca²⁺ influx generally promotes cell proliferation, migration, and cytokine production in immune cells. The anti-inflammatory cytokine, interleukin-10 (IL-10) plays a crucial role in escape from tumor immune surveillance. Recently, we discovered the dysregulated IL-10 expression and production in human T-cell lymphoma HuT-78 cells. We also showed the involvement of Smad2/3 signaling pathway in this. Both Smad2 and Smad3 were constantly activated (phosphorylated) in HuT-78 cells, Phospho-Smad2 (P-Smad2) protein expression and nuclear translocation of P-Smad2 were inhibited by the K_Ca3.1 activators. Pre-treatment with a calmodulin kinase II inhibitor, KN-62 suppressed the KCa3.1 activator-induced IL-10 down-regulation and the nuclear translocation of P-Smad2. These results suggest that the K_Ca3.1 activator-induced transcriptional repression of IL-10 in HuT-78 cells is due to the inhibition of the nuclear translocation of P-Smad2 and K_Ca3.1 activators have potential as a therapeutic option to suppress the tumor-promoting activities of IL-10 through CaMKII signaling.

Dopamine is the key neuromodulator regulating brain functions and its dysfunctions leading to various neuropsychological disorders such as Parkinson's disease and schizophrenia. Despite much interest, no versatile and safe probe is available that mimic chemical and biological activity of dopamine. Here, we report the development of the first alkyne-tagged neurotransmitter, mono-N-propargylated dopamine (MNPD) as a versatile dopamine prove. MNPD was synthesized from dopamine with five chemical reactions, and is structurally different from endogenous dopamine only by the propargyl group (–CH₂ –C≡CH) on its amino group, which suggests minimal effect on the chemical properties. Importantly, the alkynyl group allows coupling of MNPD with azide group (–N₃) containing molecules such as fluorophores by Click-Chemistry. Indeed, MNPD was incorporated into dopaminergic neuronal cell lines as well as brain tissues, was fixed at the site, and was able to be visualized by fluorescent microscopy. Furthermore, MNPD showed dopamine-like bioactivity, suggesting MNPD can serve a true biological analog probe of dopamine. In addition to these recent results, other molecule proves, MNPD derivatives, will be discussed.

Anxiety disorders represent the most prevalent mental health problem. While pharmacological and cognitive-behavioral therapies are effective for these disorders, approximately one third of patients show treatment resistance. Electroconvulsive treatment (ECT) could be a candidate treatment option for the medication-resistant anxiety disorders. However its anxiolytic efficacy and mechanism of action are poorly understood. Here we characterized a potential anxiolytic effect of ECT and investigated its neuronal basis using mice. We found that a few times of ECT produced a robust anxiolytic-like behavioral effect. Further repetition of ECT additionally induced an antidepressant-like effect. Repeated ECT also strongly enhanced serotonin 5-HT₄ receptor-dependent synaptic modulation mediated by endogenous serotonin in the hippocampus. In mice lacking the 5-HT₄ receptor, the anxiolytic-like, but not antidepressant-like, effect of ECT was significantly attenuated. These results suggest a specific involvement of the enhanced 5-HT₄-dependent neuromodulation in the anxiolytic effect of ECT. Our finding suggests that ECT can be a plausible treatment option for anxiety disorders with onset of action faster than its antidepressant effect.

Orexin and melanin-concentrating hormone (MCH) neurons innervate each other and have opposite effects for rapid eye movement sleep (REMs) regulation. Narcolepsy patients appear abnormal REMs behaviors such as rapid transitions into REMs. The causal role of orexin neurons in narcolepsy is well established, but that of MCH neurons remains unclear. We hypothesized that in the absence of orexins, the effects of MCH on REMs can be unbalanced, potentially contributing to aspects of abnormal REMs observed in narcolepsy. To test this hypothesis, we generated MCH-Cre::OX-KO mice and characterized sleep-wake behaviors and cataplexy with chemogenetic activation and pharmacological inhibition of MCH signaling.

In mice lacking orexins, activation of the MCH neurons also increased abnormal intrusions of REMs manifest as cataplexy and short latency transitions into REMs (SLREM). Conversely, a MCH receptor 1 antagonist, SNAP 94847, almost completely eliminated SLREM and cataplexy in OX-KO mice. These findings affirm that MCH neurons promote REMs under normal circumstances, and their activity in mice lacking orexins likely triggers abnormal intrusions of REMs into non-REMs and wake, resulting in the SLREM and cataplexy characteristic of narcolepsy.

The promotive effect of rehabilitation after brain stroke has been reported. However, the underlying mechanisms are not clear. The aim of this study is to reveal the effects and the mechanisms of exercise on functional recovery after cerebral ischemia. To induce reproducible permanent cerebral ischemia, male C.B-17/Icr-+/Jcl mice, aged 7weeks were subjected to middle cerebral artery occlusion (MCAO). All mice were divided into 4 groups: sham group, sham + exercise group, MCAO group and MCAO + exercise group. Exercise groups were housed in a cage with a running wheel and let free to run. Behavioral tests were performed using wire hang test and Rota-Rod test at day 14 post-ischemia. Grid walking test was performed during day 2-14 post-ischemia. Then, brains were removed, and the neuronal processes and dendritic spines of pyramidal cells in the layer 5 were visualized by microinjection with Lucifer yellow. Our preliminary results suggest that voluntary exercise can facilitate functional recovery after cerebral ischemia. We will propose the hypothesis that exercise-induced functional recovery after cerebral ischemia is related to the change of dendritic spine morphology.

in vivo Phenomic Screening System for Antiepileptic Drugs using Dravet Syndrome Zebrafish Model

Epilepsy is a common chronic neurological disease affecting almost 1 million people in Japan and 50 million people worldwide. Despite availability of more than two dozen FDA-approved antiepileptic drugs, one-third of patients fail to receive adequate seizure control. Specifically, pediatric genetic epilepsies are often the most severe, debilitating and pharmaco-resistant forms of epilepsy.

The discovery of epilepsy associated genes suggests varied underlying pathologies and opens the door for development of precision medicine for each genetic epilepsy. Over 80% of patients diagnosed with Dravet syndrome carry a de novo mutation within the voltage-gated sodium channel gene SCN1A and these patients suffer with drug resistant and life-threatening seizures. Here we have developed zebrafish models for Dravet syndrome featuring inactivation of SCN1A with an emphasis on phenomics. we will also report recent drug screening efforts using our models with a focus on assay protocols and predictive pharmacological profiles. As the discovery and development phase rapidly moves from the lab-to-the-clinic for Dravet syndrome, it is hoped that this zebrafish-based drug discovery strategy offers a platform for how to approach any genetic epilepsy.

Insulin secretion from pancreatic β-cells, which is regulated by intracellular Ca²⁺ signals, plays a crucial role in the control of blood glucose levels. Although Ca²⁺ signals in β-cells have been analyzed using in vitro/ex vivo preparations including cell lines, isolated β-cells and isolated islets, in vivo Ca²⁺ imaging has proved to be challenging. Thus, while activities of β-cells in living animals are under the influence of autonomic nervous system, hormones and other substances, Ca²⁺ signals of β-cells under physiological conditions have remained elusive. We here report an in vivo β-cells Ca²⁺ imaging method using a transgenic mouse line expressing a genetically encoded Ca²⁺ indicator YC-Nano50. Using the method, we succeeded in analyzing Ca²⁺ signals in β-cells in laparotomized mice under anesthesia, and observed synchronized Ca²⁺ oscillations in β-cells within individual islets. Furthermore, simultaneous Ca²⁺ imaging in multiple islets revealed synchronized Ca²⁺ oscillations among islets, which is the basis for pulsate insulin secretion that underlies the oscillation of blood insulin level. The present method is expected to help us gain deeper insight into the regulation of insulin secretion and diabetes.

Near-infrared (NIR) fluorescence (wavelength: 700-900 nm) is useful for in vivo imaging because of its high tissue permeability and low contamination of autofluorescence. We developed DeQODEtag system as a chemical biology tool for specific labeling of target cells using NIR fluorescence in living animals. In this system, cells expressing DeQODEtag, which is our previously obtained single-chain variable fragment (scFv) binding to a dinitrophenyl quencher, are specifically highlighted by small-molecular QODE probes consisted of a fluorophore and the quencher. We performed a chemical structure screening and obtained a high-affinity QODE probe, namely 6SiR700-pCF3oNP. A cell line with stable expression of EGFP-DeQODEtag was prepared based on a metastatic osteosarcoma cell line LM8. In a co-culture system, our probe selectively stained DeQODEtag-expressing LM8 cells against the original LM8 cells. We also prepared mouse models bearing implanted DeQODEtag-expressing LM8 cells, and performed in vivo imaging of the mice with intravenously administered QODE probes. Our developed DeQODEtag system successfully performed clear visualization of target tumor cells with NIR fluorescence in living mice. Future work will focus on applications of our system to in vivo brain Imaging.

Human audition can distinguish frequencies that are only 0.2% apart as well as perceive millionfold differences in acoustic pressure. These properties stem from sound-induced nanoscale vibrations in sensory epithelium of the inner ear. The epithelium is composed of three layers; sensory hair-cell, supporting-cell, and extracellular-matrix layers. Although each layer seem to show different vibration properties in vivo, this characteristics has not yet been precisely determined. In this study, we have developed an imaging system that can record the tomography and motion of the epithelium. The underlying technique is based on a commercial optical coherence tomography (OCT) system. We equipped the system with a powerful, broadband light source. This arrangement allows us not only to achieve depth resolution of ～1.8 μm in the tomographic image but also to pursue amplitude and phase of nanoscale vibrations in each of the three layers in a live guinea pig. The system has a potential to reveal physical networks across the three layers and thereby it may contribute to identification of a force transport mechanism underlying biomechanical amplification in the cochlea.

Audition is triggered by sound-induced nanoscale vibrations in sensory epithelium of the inner ear. The epithelium is composed of three layers; sensory hair-cell, supporting-cell, and extracellular-matrix layers. Conventional optical coherence tomography (OCT) systems planarly scan the sample to reconstitute the image and analyze the motions. However, because of the low scanning resolution, planar distribution of the vibrations inside the epithelium remains uncertain. To address this issue, we describe an advanced OCT imaging system. A high-resolution microscope and ultra-speed CMOS camera were incorporated into the system, which resulted in acquisition of a square image of 0.5 x 0.5 mm at once with resolution of 2 μm. A supercontinuum broadband light source in the system achieved depth resolution of 1.8 μm. A vibrometric technique that can stroboscopically capture the motion permits us to detect vibrations of up to 30 kHz in the object. Through a microscope equipped with the system, we recorded a planar distribution of nanoscale vibrations on the extracellular-matrixlayer in a live guinea pig.This system may contribute to clarification of a fundamental mechanism underlying hearing.

Objective: Some histamine H₁ receptor (H₁R) antagonists have sedative effects, caused by the blockade of histamine neural transmission. Desloratadine is a newly-marked antihistamine, but its sedative properties have not been examined by positron emission tomography (PET). We examined the brain H₁R binding potential ratio (BPR), H₁R occupancy (H₁RO) and the subjective sleepiness after oral administration of desloratadine and loratadine, the prodrug of desloratadine.

Methods: Eight healthy male volunteers underwent PET imaging with [¹¹C]doxepin after single oral administration of desloratadine (5 mg), loratadine (10 mg), or placebo in a double-blind crossover study. BPRs and H₁ROs in the cerebral cortices were calculated. Subjective sleepiness was quantified by the LARS and the SSS.

Results: BPR after loratadine administration was significantly lower than placebo (p<0.05), but BPR after desloratadine was not significant. There was no significant difference, however, between H₁RO after desloratadine and loratadine administration. The subjective sleepiness was not significantly different among the two antihistamines and placebo.

Conclusion: At therapeutic dose, desloratadine did not bind significantly to brain H₁Rs and did not cause significant sedation.

Suppression of Defender against cell death 1 leads to cardiomyocyte death

Background/Purpose】Since cardiomyocytes lose proliferative capacity soon after birth, inhibiting cardiomyocyte death is one of the important therapeutic strategies for heart failure. The goal of this study is to identify novel genes which inhibit cell death in cardiomyocytes (CMs).

【Method/Results】We found a CM death suppressing gene Dad1, whose function in the heart has not yet been elucidated. The siRNA was used to suppress the expression of Dad1 in neonatal rat CMs (NRCMs) and evaluate cell viability. As a result, suppression of Dad1 induced cell death. In addition, suppression of Dad1 increased GRP78 expression, which is an ER stress marker, suggesting ER stress is involved in cell death. In contrast, apoptosis inhibitor z-VAD-fmk failed to suppress cell death caused by suppression of Dad1. Moreover, the expression of Stt3a, a catalytic subunit of the oligosaccharyltransferase (OST) complex which Dad1 stabilizes, was suppressed, and cardiomyocyte death was also induced by suppression of Stt3a expression.

【Conclusion】 Dad1 inhibits NRCM death and ER stress and stabilizes OST complex. Thus, enhancement of Dad1 expression could be a new therapeutic target for heart failure.

Comprehensive search for cardiomyocyte death-related genes using forward genetic screening

[Background] Cardiomyocyte (CM) death is an important risk factor in heart failure. Though molecular mechanisms of apoptosis have been well studied, those of necrosis remain to be fully elucidated. To explore novel genes related to non-apoptotic cell death induced by reactive oxygen species, we attempt genome-wide and forward genetic screening using lentiviral shRNA library. For this purpose, we optimized experimental conditions using neonatal mouse CMs.

[Methods] After infection with a pool of lentiviral shRNA library followed by the treatment with or without H2O2 (100 μM), we prepared genomic DNA from neonatal mouse CMs. The sequence of shRNA in the lentiviral vector was analyzed using the next generation sequencer.

[Results] We determined 5359 shRNA sequences targeting approximately 1000 genes. Focusing on difference of reads between 2 groups, 12 candidate genes were obtained. 2 candidates were knocked down in neonatal mouse CMs, but cell death was not suppressed.

[Conclusions] Genome wide lentiviral shRNA screening in cardiomyocytes can be useful for determining novel genes related to cell death. However there are still some problems to be solved because of the low infection efficiency in CMs.

β_1-adrenergic receptor (β₁AR) is a potent regulator of cardiac function. Previously, we showed that recycling endosome (RE) is key organelle of the intracellular trafficking of β₁AR induced by ligand stimulus. Here, we found that HACE1, an E3 ligase, is localized in RE and ubiquitinates β₁AR internalized by the ligand isoproterenol (Iso). Silencing of HACE1 by siRNA resulted in a decline of β₁AR on the cell surface at steady state. Given that the ubiquitinated Rab11 functions as a recycling factor, ubiquitinated Rab11 mediated by HACE1 might recycle β₁AR to the cell surface. Ubiquitination of β₁AR was increased by Iso treatment, which was further enhanced with either bafilomycin A₁(BafA₁), a lysosome inhibitor or MG132, a proteasome inhibitor. β₁AR was co-localized with Rab12 at RE and lysosome and interacted with each other. The co-localization at lysosome was strengthened in the presence of BafA₁, and the interaction was enhanced by Iso stimulus. Silencing of Rab12 led to stabilization of β₁AR with or without Iso. These data suggest that Rab11 and Rab12 were involved in the recycling and degradation of β₁AR through ubiquitination of Rab11and β₁AR mediated by HACE1 in RE, respectively.

Aim: Proarrhythmic potential of donepezil was analyzed. Methods: Exp.1: We infused 0.1 and 1 mg/kg, i.v. of donepezil hydrochloride to the chronic atrioventricular block dogs (n=4). Exp.2: We infused 0.01, 0.1 and 1 mg/kg, i.v. of the drug to the halothane-anesthetized dogs (n=4). Results: Exp.1: Low dose did not alter the variables. High dose increased ventricular rate, and induced non-sustained VT in 2 animals at 14-16 min, but TdP was not observed. Exp.2: Low dose did not affect the variables. Middle dose prolonged HV and QRS without altering the other variables. High dose increased heart rate, mean blood pressure, cardiac output, ventricular contraction, but decreased the preload to left ventricle and total peripheral resistance. The dose delayed the repolarization for 5-10 min, prolonged JT_peakc for 5-30 min, T_peak-T_end at 5 min, HV for 5-15 min and refractory period at 5 min, but shortened AH at 15 and 30 min. Conclusion: The high dose would have increased adrenergic tone, indirectly stimulating Ca²⁺ channel, which may explain its effects on JT_peakc and AH, whereas it may also directly inhibit Na⁺ and K⁺ channels, increasing ventricular refractoriness. Thus, temporal discordance between direct and indirect actions may well explain proarrhythmic profile of donepezil.

Introduction: TdP occurred in LQT3 patient after switching perospirone (PER) to blonanserin (BLO) for her nocturnal delirium. PER blocked α₁-adrenoceptors with pA₂ of 2.0 ng/mL. While BLO may also have such potential, 1 μg/mL of it can inhibit hERG K⁺ channel by 82%. We studied onset mechanism of TdP.

Methods: PER hydrochloride (n=4) and BLO (n=4) of 0.01, 0.1 and 1 mg/kg, i.v., were administered to the halothane-anesthetized dogs.

Results: The low dose of PER decreased total peripheral vascular resistance (TPR), but increased heart rate (HR) and cardiac output (CO), and facilitated AV conduction. The middle dose decreased mean blood pressure (MBP) and prolonged repolarization period besides those observed after the low dose. Although the high dose decreased MBP and TPR, it did not increase HR or CO. It tended to delay AV conduction, and significantly prolonged repolarization period. BLO dose-dependently decreased TPR, but increased HR, CO and cardiac contractility without affecting electrophysiological variables.

Conclusion: Lack of the reflex-mediated increase of sympathetic tone after the high dose of PER may suggest its Ca²⁺ channel inhibition suppressing TdP. BLO-induced increase of Ca²⁺ current might have induced TdP in the LQT3 patient.

Roles of macromolecular complexes in calcium-sensitivity of the cardiac I_Ks channel

Cardiac repolarization process is regulated by the slow component of the delayed rectifier potassium (I_Ks) channel composed of Ca²⁺-sensitive KCNQ1 and KCNE1. Although calmodulin and AKAP9 regulate I_Ks channel for instance, the entire macromolecular system has not been elucidated. Thus, to seek macromolecular complexes comprehensively, we performed proteomics analysis using a transgenic mouse overexpressing KCNQ1-KCNE1 fusion protein specifically in the heart. We identified 163 proteins as potential I_Ks binding partners by immunoprecipitation using a goat anti-KCNQ1 antibody. Pathway analysis of these proteins revealed that the most significant biofunction is the Calcium Signaling including 16 molecules. To understand how the protein-protein interactions affect Ca²⁺-dependent I_Ks regulation, we picked up the cardiac Na⁺-Ca²⁺ exchange transporter (NCX1). Pull-down assays in dog ventricles with purified GST-fusion proteins of full-length or parts of KCNQ1 identified that the N- and a proximal C-terminus of KCNQ1 contribute the interaction. These results imply that the Ca²⁺-sensitivity of I_Ks channel is regulated by the interaction between the I_Ks channel and NCX1. Pharmacological experiments will be performed to pursue its mechanistic insights.

The excellent tumor selectivity of amino acid-based imaging probes, [¹⁸F]FAMT (3-[¹⁸F]fluoro-a-methyl-l-tyrosine) and [¹²³I]IMT (3-[¹²³I]iodo-a-methyl-l-tyrosine), is supported by their selectivity for LAT1 (L-type amino acid transporter 1) which is specifically expressed in tumor cells. However, FAMT and IMT show a physiological background in kidney. Their renal accumulation is suppressed by probenecid, suggesting a contribution of organic anion transporter OAT1. To reveal the critical moieties responsible for the interaction with OAT1, we performed a structure-activity relationship analysis of aromatic amino acid derivatives in vitro. We revealed that both halogen and hydroxyl groups on the benzene ring are critical for the interaction with OAT1. Their positions on the benzene ring also affected the interaction. In contrast, the a-methyl moiety, which is essential for the selectivity to LAT1, was dispensable for the interaction with OAT1. These results hold a significant implication for designing not only the tumor-specific imaging probes with less renal background, but also the therapeutic agents for targeted radionuclide therapy with less adverse renal damage.

Idiopathic pulmonary fibrosis(IPF) is considered a fatal respiratory disease. However, a large number of anti-fibrotic drugs described in the current experimental models including bleomycin(BLM)-induced fibrosis have not been translated into clinical practice successfully, suggesting that a new pulmonary fibrosis model mimicking most of pathological features of human IPF is needed. We established a new pulmonary fibrosis mouse model by injecting FeCl₃ solution into the left upper lobe central part. In our lung lobe-specific fibrosis model, fibrogenesis was progressive and irreversible, the feature of which mimics human IPF and cannot be observed in other lung fibrosis models. Here, we investigated how ferric chloride could induce pulmonary fibrosis.

　At 10 days post-injury with ferric chloride, severe fibrosis of the whole lobe was observed. We temporally and spatially followed the injury process of the lung by monitoring the indices such as ferrous iron accumulation, production of Reactive oxygen species , endoplasmic reticulum stress and apoptosis. We also performed a comprehensive analysis of microRNAs expression. We will discuss the molecular mechanisms underlying pulmonary fibrosis induced by ferric chloride, compared with BLM-induced fibrosis model.

Reducing pancreatic β cell failure may suppress the onset and progression of type 2 diabetes. Endoplasmic reticulum stress (ER stress) has been reported to be involved in the onset of metabolic diseases such as diabetes and obesity. We previously shown that flurbiprofen, a nonsteroidal anti-inflammatory drug, may have chaperone activity and can suppress ER stress. In this study, we investigated possibility that flurbiprofen may ameliorate diabetes through reducing ER stress-induced pancreatic β cell death. We found that flurbiprofen suppressed the ER stress-induced expression of C/EBP homologous protein (CHOP), an apoptotic transcription factor, in mouse pancreatic β cells (Min6 cell line). Additionally, flurbiprofen suppressed ATF4, an upstream regulator of CHOP, suggesting that flurbiprofen may protect β cells by suppressing apoptosis through regulating ATF4-CHOP pathway. Furthermore, we found that flurbiprofen reduced blood glucose levels, and increased pancreatic and serum insulin levels without affecting body weight in db/db diabetic mice model. We are now performing comprehensive analysis using microarrays to further elucidate pharmacological action of flurbiprofen in pancreatic β cells. Overall, flurbiprofen may be able to ameliorate diabetes by reducing ER stress in β cells.

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol (DAG) to phosphatidic acid. We have previously demonstrated that DGKδ acts as a negative regulator of β-cell proliferation. However, it is still unknown whether DGKδ deficiency in pancreatic β-cells alleviates diabetes. In the present study, we investigated the effect of β-cell specific DGKδ deficiency on hyperglycemia in streptozotocin (STZ)-induced diabetic mice. We administered STZ to 5-wk-old mice and performed measurement of body weight and blood glucose level for 60 days. At 11 days after STZ administration, there was no significant difference in serum insulin concentrations, pancreatic insulin content, or β-cell area between β-cell specific DGKδ knockout (βDGKδKO) and control mice. At 60 days, in contrast, blood glucose level was significantly lower and serum insulin level and pancreatic insulin content were significantly higher in βDGKδKO mice than in control mice. In morphological analysis, a significant increase in β-cell area was observed in βDGKδheteroKO mice compared with control mice. These results suggest that DGKδ deficiency in pancreatic β-cells prevents STZ-induced hyperglycemia, which is likely due to maintained β-cell mass resulting from promoted β-cell proliferation.

Diabetic neuropathy (DN) is one of the most common complications of diabetes, and has a prevalence of as high as 50% in diabetic patients. Allodynia and hyperalgesia are early symptoms of the disease. Sensory paralysis is a late symptom, and is frequently accompanied by a poor prognosis, including limb amputation. However, there is currently no effective treatment for DN. In the present study, we evaluated the effect of a bioactive natural product in a mouse model of DN produced by injection of 200 mg/kg streptozotocin (STZ). The bioactive natural product, 30 mg/kg, was administered to the diabetic mice for a period of 3 weeks, starting 1 week after the STZ injection. The behavioral effects of the bioactive natural product were evaluated using the von Frey test (to assess mechanical allodynia) and the hot plate test (to assess thermal hyperalgesia). Furthermore, we examined conduction velocity and blood flow in the sciatic nerve. Treatment with the bioactive natural product ameliorated the allodynia and hyperalgesia, and it increased conduction velocity and blood flow in the sciatic nerve in the diabetic mice with DN. These findings suggest that the bioactive natural product has therapeutic potential for the treatment of DN.