Inflammation and immune responses are evoked locally by invasion and accumulation of immune cells into the lesion sites. The cell trafficking of immune cells into the tissue from the blood is closely regulated by a number of cell adhesion molecules and chemotactic factors including chemokines.

Fractalkine (FKN)/CX3CL1 is a membrane-bound chemokine possessing a chemokine/mucin hybrid structure and has a dual function as an adhesion molecule and a chemoattractant. FKN is mainly expressed on activated endothelial cells and its cognate receptor, CX3CR1, is expressed on cytotoxic effector lymphocytes and monocytes/macrophages. To date, a lot of important roles of the FKN-CX3CR1 axis has been identified: (1) the rapid capture and firm adhesion, (2) chemotaxis, (3) the enhancement of the transmigration, (4) the patrolling behavior of monocytes, (5) the accessory cell function and (6) the cell survival.

In this symposium, we will overview the pathological roles of the FKN-CX3CR1 axis in several inflammatory and autoimmune disease models revealed by anti-FKN mAb, and its distinct mode of action from other cytokine inhibitors.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by inflammation and joint destruction that causes significant morbidity and mortality. However, the combined use of methotrexate and biologics targeting TNF and IL-6 has revolutionized treatment of RA. Clinical remission is now realistic targets, achieved by a large proportion of RA patients, and rapid and appropriate induction of remission by the intensive treatment is prerequisite to halt joint damage and functional disabilities. Recently, orally available small molecules targeting Janus kinase (JAK) has taken in the therapeutic armamentarium in RA. Such a progress in treatments with biologics and JAK inhibitors of RA is now applied for various rheumatic diseases and systemic autoimmune diseases. Furthermore, treatment holiday of biologics is feasible in some patients with RA after maintaining deep remission by the intensive treatments, which has the potential of reducing adverse effects and medical costs as well as approaching to immunological remission. Finally, we currently try to treat patients with different biologics targeting TNF, IL-17 and IL-12/IL-23 based on the difference of lymphocyte phenotype. Such a treatment strategy should be guided by molecular, cellular and/or immunological mechanisms and a systematic approach to design a precision medicine to rheumatic diseases should help to achieve the goal.

The development of cancer immunotherapy is the history of activation of its immune system, however it is also the history of failure of the development of the therapy. In these histories, the concept of immune checkpoint was born resulting a big breakthrough. While many excellent fundamental researches have not reached clinical application, checkpoint inhibitors succeeded in translational research and drug discovery. Since anti-PD-1/PD-L1 blockade therapy has been delivered to the world, types of tumor were divided into two groups. One is sensitive type by immunotherapy called as Hot tumor, and the other one is the other way called as Cold tumor which is difficult to demonstrate effectiveness. In recent years it has become clear that the gut microbiota has a great influence in terms of tumor immune microenvironment. We are actively pursuing analysis of gut microbiota with cases of treatment by cancer immunotherapy and chemotherapy. The development of this research is boosted by improvement of the next generation sequencer and analysis method. In addition to this, we also add non-linear analysis by adding Artificial Intelligence (AI) to analytical methods. These results will be used for future fecal transplantation as a translational research. We introduce our new findings, explain the trend of the development of cancer immunotherapy in the world, and we will state what kind of action we are taking for it.

Some infected with human T-lymphotropic virus type 1 (HTLV-1), which causes adult T cell leukemia/lymphoma (ATL), develop the neuroinflammatory disease HTLV-1 associated myelopathy (HAM). Suffering from progressive spinal cord paralysis, HAM patients experience a low quality of life with high unmet needs. Since the prognosis for HAM patients is extremely poor, there is a strong demand for a novel therapeutic strategy. Therefore, we established a national patient registration system (HAM-net) in collaboration with patient groups to gather data from and distribute information to patients on a nation-wide scale. By establishing a registration system for HAM patients, we have learned more about the variation in the rate of HAM progression that enables to divide patients into groups based on progression speed and compare attributes between groups. In addition, we recently showed that HTLV-1 mainly infects CCR4+ T-cells and causes functional abnormalities that are believed to drive HAM pathogenesis. We next demonstrated that anti-CCR4 antibody is effective at reducing the proviral load and inflammatory response in PBMCs from patients with HAM. Given the overwhelming evidence to support the idea that anti-CCR4 antibody could benefit HAM patients, we began an Investigator-led clinical trial. The phase 1/2a trial of the anti-CCR4 antibodies proceeded smoothly; in January 2016, the clinical studies were completed, and a proof of concept of the safety and efficacy of the treatment was obtained (N Engl J Med, 2018).

Bile salt export pump (BSEP, encoded by ABCB11), an ABC transporter localized on the canalicular membrane (CM) of hepatocytes, mediates biliary excretion of bile acids (BA). Its dysfunction impairs bile formation, a liver condition called intrahepatic cholestasis (IC). PFIC2, the most severe form of IC caused by mutation in ABCB11, progresses to liver failure and death before adulthood. Currently, the only therapeutic approach is liver transplantation.

We have shown that PFIC2-causing mutations predominantly affect expression of BSEP on the CM but not its transport activity and then searched potential compounds to restore BSEP expression. Sodium 4-phenylbutyrate (NaPB), a drug approved for urea cycle disorder (UCD), was found as the candidate. Animal experiments and retrospective study in UCD patients indicated that treatment with NaPB increases BSEP expression on the CM and thereby its function. Clinical study in three PFIC2 patients showed that NaPB therapy markedly improved biochemical tests, clinical symptoms, and liver histology.

Based on these facts, we have started clinical trial to obtain approval for new indications of NaPB for PFIC2 (UMIN000024753) and clinical study to investigate therapeutic potency of NaPB in patients with IC other than PFIC2 (UMIN000027666).

Recent advances in super-resolution microscopy have overcome a limitation ofspatial resolution in fluorescence imaging imposed by the diffraction of light,enabling nanoscale molecular imaging with conventional fluorescent probes.Single-molecule localization based super-resolution microscopy, includingstochastic optical reconstruction microscopy (STORM), provides multi-color andthree-dimensional imaging with a remarkable spatial resolution, and thusresolves detailed nanostructures in biological specimens. Fortunately, it can beperformed with a simple wide-field microscope setup and is completely compatiblewith immunohistochemical fluorescent staining methods. In this seminar, I willtalk a detailed method for STORM imaging, from sample preparation to imageanalysis, with some tips on how to improve the spatial resolution. I will thenshow some examples for STORM imaging of immunohistochemical specimens, includingthe brain and pancreas.

Recent advances in super-resolution microscopy has enabled diffraction-unlimitedimaging of cellular structures and protein distributions in living cells. Manycommercially available microscopes are now equipped with super-resolution mode,however, knowledge about how to choose an appropriate microscopy for eachexperiment is not shared among researchers yet. In this session we willintroduce various types of state-of-art super-resolution microscopes with theiractual applications and discuss about some critical points to notice whenchoosing microscopes for live-cell super-resolution imaging. We would also talkabout our recent development of a novel live-cell RNA imaging tool and itsapplication with super-resolution microscopy.

Neuropathic pain associated with cancer, diabetic neuropathy, chemotherapy or nerve trauma is an intractable chronic pain characterized by mechanical allodynia and abnormal pain hypersensitivity evoked by innocuous stimuli. Unfortunately, this disorder has no specific treatment. To discover potential new pain medications, I am merging high-throughput screening technologies with a drug discovery strategy that seeks new effects of approved drugs known as "drug repositioning". In this seminar, I will show that by using high-throughput Ca²⁺ imaging instrument, the compound duloxetine (a serotonin-norepinephrine reuptake inhibitor) inhibits the function of the purinergic receptor P2X4 (a subtype of ATP-gated non-selective cation channels), which is a potential therapeutic target for treating neuropathic pain. In addition, by using a newly established in vitro high-throughput phenotypic assay, we have discovered that fulvestrant (a drug for treatment of postmenopausal women with advanced breast cancer) exhibits a protective effect on oxaliplatin-induced neuronal damage and allodynia.

Observing neuronal activity of free-moving animals gives us a lot of information that helps us to understand the neural computations that mediate behavior. While many techniques have been utilized to measure neuronal activity in specific brain regions, these regions are made up of genetically and anatomically heterogeneous sub-populations, and dissection of neuronal activity of interest is still an ongoing challenge.

Neural activity causes rapid changes in intracellular free calcium. The development of genetically encoded calcium indicators, such as GCaMPs, allows us for in vivo visualization of calcium dynamics of specific neuronal populations. In this seminar, I will outline recent advances in calcium imaging technology, including fiberphotometry, and show our recent works on neural circuit mechanisms controlling appetitive and aversive learning.

Although Kir6.2, one of the subunits composed ATP-sensitive potassium (K_ATP) channels, are widely distributed in the brain, the roles in emotional behavior are not yet fully understood. Here we investigated the behavioral characteristics of Kir6.2-deficient (Kir6.2^-/-) mice. Kir6.2^-/- mice showed anxiety-like behavior in the elevated-plus maze test and light-dark test, especially, it was prominent in females. Immunohistochemical studies showed that Kir6.2 was co-localized with tryptophan hydroxylase (TPH) in the dorsal raphe nuclei and tyrosine hydroxylase (TH) in the ventral tegmental area/locus coeruleus. Interestingly, TPH expression in the midbrain was significantly elevated in female Kir6.2^-/- mice. These results suggest that Kir6.2 expressed in serotonergic neurons could play a key role in emotional behavior. Furthermore, we investigated whether pharmacological blockade of K_ATP channels affects the emotional behavior. Mice that had been injected intracerebroventricularly with glibenclamide, a selective K_ATP channel blocker, showed anxiety-like behavior in the elevated-plus maze test. These results confirm a critical role of K_ATP channels in regulation of emotional behavior.

Depression is the major mental disorder and over one million patients are suffering from this disease. It was also reported that the number of patients showing resistance toward anti-depressant, i.g. SSRI and SNRI, got increase. We have already known that molecular mechanism underlying depression is heterogeneous so that it is hard to estimate the efficacy of anti-depressant without molecular rationale. Postmortem human brain analysis indicated that the number of AMPA receptors (AMPARs), major molecule controlling synaptic functions, varied among depression patients and the results of these analysis were not consistent. To clarify the dynamics of AMPARs in depression patients, we developed the novel PET imaging method to measure the density of AMPARs in depression patients. This result showed that depression patients decreased AMPARs broadly throughout the brain. This fact motivated us to develop novel AMPARs potentiator in order to cure the depression. To find the compound showing high affinity to AMPARs and high BBB penetratability, we modified the compound named PEPA, already known to bind specifically to AMPARs, and finally succeeded in synthesizing the seed compound. This compound could exert the anti-depressant effect quickly and sustained for a week after the cessation of drug administration. Furthermore, this anti-depressant effect was significantly stronger that another AMPARs potentiators.

Desmosome is a cell structure of cell-to-cell adhesion in epithelia and cardiac muscles. Desmoplakin, a component of desmosome, is highly expressed in the dentate gyrus (DG) in the brain, although there is no evidence for desmosome structures in neurons. We previously found that the expression of desmoplakin in mouse DG was decreased by antidepressant treatments, including selective serotonin reuptake inhibitors (SSRIs) and electroconvulsive stimulation (ECS), a model of electroconvulsive therapy. However, the role of desmoplakin in hippocampus has been unknown. In this study, we generated adeno associated virus expressing artificial microRNA targeting desmoplakin to suppress desmoplakin expression, and injected it into the mouse DG. We conducted anxiety-like and depressive-like behavioral tests, and examined synaptic potentiation and mature markers expression. Knockdown of desmoplakin in the DG did not affect anxiety-like and depressive-like behaviors. We further found that desmoplakin knockdown decreased synaptic potentiation and increased the mature marker expression in the DG. These results implicate that down-regulation of desmoplakin expression may oppositely affect antidepressant effects in the hippocampus.

Chronic cerebral hypoperfusion (CCH) is manifested in a various CNS diseases, including neurodegenerative and mental disorders accompanied by cognitive impairment. We previously demonstrated that microglial activation via TRPM2, a Ca²⁺-permeable non-selective cation channel, induced excessive inflammatory responses, white matter injury, and resultant aggravation of cognitive impairment in a mouse CCH model with bilateral common carotid artery stenosis (BCAS), while there was no direct evidence on the contribution of microglia. To clarify the role of microglia in the BCAS model, we used PLX3397, an orally-active inhibitor of colony-stimulating factor 1 receptor (CSF1R), since microglia require CSF1R signaling for survival. When mice were fed with PLX3397-containing chow at 290 mg/kg for 3 weeks, virtually all Iba1-immunopositive microglia were eliminated from the brains, without obvious deficits in the behaviors. When the mice were then subjected to BCAS, white matter injury and cognitive dysfunction at additional 28 days were improved in the PLX3397-fed mice compared with control mice. These results suggest that microglia play destructive roles in the development of CCH-induced cognitive impairment.

Chronic itch is a major symptom in various skin diseases, such as atopic and contact dermatitis, but its mechanism remains to be determined. We have previously shown that spinal dorsal horn (SDH) astrocytes become activated in mouse models of chronic itch and that astrocyte-derived lipocalin-2 (LCN2) is crucial for maintaining chronic itching. However, how LCN2 enhances spinal itch neurotransmission is not understood. In this study, using Gastrin-releasing peptide receptor (Grpr)-egfp mice to label itch-specific neurons in SDH, we found GRP-induced depolarization of excitatory GRPR⁺ neurons was greatly potentiated in contact dermatitis model mice. Genetic inhibition of signal transducer and activator transcription 3 (STAT3) in SDH astrocytes ameliorated chronic itch and also normalized enhancement of GRP-induced depolarization of excitatory GRPR⁺ neurons in chronic itch model. Furthermore, coadministration of LCN2 and GRP also potentiated GRP-induced depolarization of excitatory GRPR⁺ neurons. Our finding indicate that reactive astrocytes facilitated SDH itch transmission in chronic itch via upregulated LCN2.

A pathogenic role for high-mobility group box 1 (HMGB1) protein has been postulated in severe sepsis. Histidine-rich glycoprotein (HRG) is a 75-kDa plasma protein which was recently proposed as a new biomarker to predict the outcome of sepsis patient and was demonstrated to improve the survival of septic mice through the regulation of neutrophils and vascular endothelial cells. Here, we monitored the effects of HRG on the lipopolysaccharide (LPS)-mediated release of HMGB1 and the HMGB1-mediated modulation of proinflammatory responses in EA.hy 926 endothelial cells. Our results show that LPS induced significant HMGB1 translocation from nucleus to cytoplasma and large amount of HMGB1 release from EA. hy 926 endothelial cells which effectively inhibited by HRG. Furthermore, HRG potently inhibited high expression of adhesion molecules and release of inflammatory cytokines from HMGB1-activated endothelial cells. HMGB1 up-regulated proinflammatory responses by interacting with three pathogen-related pattern recognition receptors: TLR2 and TLR4 and RAGE. HRG also down-regulated the cell surface expression of all three HMGB1 receptors in endothelial cells. The protective effects of HRG in severe sepsis may partially be mediated through the inhibition of HMGB1 translocation/release.

Sepsis is regarded as a gene-related disorder. Growing evidence suggests that STAT3 is a master transcriptional factor that plays an important role in inflammation. In this study, we examined whether in vivo introduction of STAT3 decoy oligodeoxynucleotides (ODNs) can provide benefits in mice with cecal ligation and puncture (CLP)-induced sepsis to assess the potential role of STAT3 in sepsis-associated organ dysfunction. Activation of STAT3 greatly increased in each of major organs after CLP in time dependent manner. We confirmed that STAT3 decoy ODNs were effectively delivered into tissues of septic mice in vivo by preparing into a complex with atelocollagen given 1 h after CLP. When STAT3 decoy ODNs were given to septic mice, abnormal production of pro-inflammatory and chemotactic cytokines was significantly reduced, histopathologic changes in lung, liver, and kidney tissues were markedly improved, and led to a significant survival advantage in mice after CLP. The STAT3 inhibitor stattic mimicked the beneficial effects of STAT3 decoy ODN transfection in septic mice. These results suggest that STAT3 is a potential therapeutic target for sepsis-associated multiple organ injury.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous population of immunosuppressive myeloid progenitor cells. We have previously shown that MDSCs are increased and play protective role in OVA-sensitized mice lung. On the other hand, glucocorticoid is the most commonly used anti-inflammatory drug for asthma. The aim of this study is, therefore, to define the potential role of MDSCs in anti-asthmatic effect of glucocorticoid. Dexamethasone (DEX) enhanced differentiation from bone marrow cells into MDSCs under co-stimulation of IL-6/GM-CSF in vitro, which was inhibited by mifepristone, a glucocorticoid receptor antagonist. DEX-treated MDSCs showed potent inhibitory effect of T cell proliferation. Consistent with in vitro results, administration of DEX significantly increased a population of MDSCs, accompanied by resolution of inflammation in lung of OVA-sensitized mice. By contrast, inhibition of MDSCs attenuated the effect of DEX. Taken together, these data reveal a novel role of MDSCs in anti-inflammatory effect of glucocorticoid and further implicated pharmacological targeting of MDSCs as a potential therapeutic strategy in asthma.

Lymphatic vessels in the diaphragm are essential for draining peritoneal fluid during peritonitis. Calcitonin gene-related peptide (CGRP), which is released from the sensory nervous system, promotes wound-induced lymphangiogenesis via receptor activity-modifying protein 1 (RAMP1), a subunit of the CGRP receptor. In this study, we examined the functional role of RAMP1 in inflammation-induced lymphangiogenesis in the diaphragm. RAMP1-knockout mice (RAMP1 KO) or their wild-type counterparts (WT) were intraperitoneally injected with LPS. Compared with WT, RAMP1 KO exhibited less lymphangiogenesis associated with reduced expression of VEGF-C and VEGF-D and with enhanced expression of Th1-related cytokines including TNF and IFN. The numbers of CD4+ cells in WT were greater than those in RAMP1 KO. RAMP1 was expressed in CD4+ cells, which also expressed VEGF-C and VEGF-D. Isolated splenic CD4+ cells stimulated with LPS enhanced expression of VEGF-C and VEGF-D in a RAMP-1 dependent manner. Deletion of CD4+ cells with an anti-CD4 antibody suppressed lymphangiogenesis. Functional assays with an intraperitoneal injection of fluorescein isothiocyanate (FITC) revealed delayed peritoneal fluid drainage in diaphragm of WT as compared with RAMP1 KO. These results suggest that RAMP1 signaling in T cells plays a critical role in LPS-induced lymphangiogenesis and lymphatic dysfunction in the diaphragm.

Virus infection is generally associated with virus-driven hijacking of host cellular machineries including translational regulation. Host shutoff is known as a strategy used by viruses to repress cellular mRNA translation and parallelly allow the efficient translation of viral mRNA. Host shutoff could be achieved by two complementary mechanisms: either direct co-opting of the translation machinery that forces better translation of viral mRNAs compared to their host counterparts, or viral-induced degradation of host mRNAs. However, it remains unclear how host shutoff and viral mRNA translation is regulated upon influenza virus infection. In the present study, to systemically analyze the dynamic changes in host and viral mRNA translation machinery, we performed ribosome profiling and RNA sequencing (RNA-seq) analysis in the mouse embryonic fibroblasts infected with influenza A (H1N1/PR8) virus. We will report the global profiles and characteristics of gene expression and translational states for host and virus mRNA during the course of influenza infection, which could lead to a better understanding of mRNA translation machinery in influenza virus infection.

Since a sustained higher excitability in the superficial dorsal horn is considered to be a crucial factor leading to chronic pain, normalizing excitatory and inhibitory balance in the dorsal horn by inhibiting GABA transporters (GAT1 and GAT3) is a promising therapeutic strategy for pain relief. However, synaptic mechanisms underlying the analgesic effects of GAT inhibitors remain unknown. Using spinal slice preparations from adult mice, we previously demonstrated that the GAT1 inhibitor NNC-711 decreases the frequency of miniature EPSCs (mEPSCs) in the dorsal horn neurons via presynaptic mechanisms including the activation of GABA_B receptors. In the present study focusing on GAT3 inhibition, we found that the GAT3 inhibitor SNAP-5114 suppressed monosynaptic C-fiber-evoked EPSCs, which was not observed in the presence of the GABA_B receptor antagonist CGP55845. By contrast, A-fiber-evoked EPSCs were less suppressed, and the frequency and the amplitude of mEPSCs were not altered. Thus, although endogenously increased GABA after blockade of GAT1 and GAT3 acts on GABA_B receptors, GAT1 and GAT3 inhibition depresses excitatory neurotransmission from spinal intrinsic interneurons and the primary afferent fibers, respectively.

Astrocytes play essential roles for the modulation of neural networks, but also have critical roles for the pathogenesis of neural disorders. Recently, we revealed that astrocytes in the primary somatosensory cortex (S1) have a critical role for neuropathic pain after partial sciatic nerve ligation (PSNL). In brief, PSNL generated Ca²⁺ excitation in S1 astrocytes, induced synaptogenesis, thereby resulting in cross-wiring of innocuous and nocuous circuits. Reappearance of mGluR5 correlated with neuropathic pain, but we still do not know whether astrocytic mGluR5 is required or not. Here, we show that mGluR5 in S1 astrocytes is a cause of cortical rewiring and neuropathic pain. Firstly, we found that mGluR5 is almost absent but reappeared in S1 astrocytes after PSNL. Secondly, we made brain astrocyte-specific mGluR5 knockout mice (astro-mGluR5-KO) and validated them. PSNL-induced mechanical allodynia was abolished in astro-mGluR5-KO mice, suggesting that upregulation of mGluR5 in S1 astrocytes should be required for mechanical allodynia. Third, mechanisms underlying astrocytic mGluR5-mediated allodynia were; (1) increase in Ca²⁺ activity, (2) expression of synaptogenic molecules such as glypican4 and hevin, (3) synaptogenesis, (4) persistent rewiring of incorrect S1 circuits. Hence, we conclude that astrocytic mGluR5 is a crucial molecule that triggers synaptogenesis in S1 after PSNL, which is the causative event for the pathogenesis of neuropathic pain.

The anti-cancer drug oxaliplatin frequently causes peripheral neuropathy. Commonly described neuropathic symptoms include aberrant sensations such as mechanical allodynia (hypersensitivity to normally innocuous stimuli). Although oxaliplatin neuropathy is a dose-limiting toxicity, preventive strategies against its side effects have not been established. We screened several sets of small-molecule chemical libraries (more than 3,000 compounds in total) using a newly established in vitro high-throughput phenotypic assay, and identified fulvestrant, a clinically approved drug for the treatment of breast cancer in postmenopausal women, as having a protective effect on oxaliplatin-induced neuronal damage. Furthermore, using a rat model of oxaliplatin neuropathy, we demonstrated the in vivo efficacy of fulvestrant to prevent oxaliplatin-induced axonal degeneration of the sciatic nerve and mechanical allodynia in histological and behavioural analyses. Thus, our findings reveal a previously unrecognised pharmacological effect of fulvestrant to prevent oxaliplatin-induced painful peripheral neuropathy and may represent a novel prophylactic option for patients receiving oxaliplatin chemotherapy.

We have shown a crucial role of HMGB1 in chemotherapy-induced peripheral neuropathy (CIPN). To clarify risk factors for CIPN, we retrospectively analyzed the clinical data of cancer patients undergoing oxaliplatin (OHP) treatment, and then studied the underlying mechanisms using a mouse model for CIPN. Analyses of 150 outpatients treated with OHP in Seichokai Fuchu Hospital identified a significant correlation between the severity of CIPN and plasma ALT, a marker of hepatic disorders. In mice, i.p. OHP at 5 mg/kg caused mechanical allodynia, which was prevented by an anti-HMGB1 antibody (AB) or soluble thrombomodulin (TM) capable of inactivating HMGB1. CCl₄ (1%, 5 ml/kg, i.p.) or ethanol (25%, 20 ml/kg x 3 for 2 days, p.o.) significantly increased ALT levels and tended to elevate HMGB1 levels in plasma. CCl₄ (every 2 days, 3 times) or ethanol (twice a day, 12 times) did not alter nociceptive threshold in naïve mice, but caused remarkable allodynia in the mice treated with OHP at 1 mg/kg, a subeffective dose, which was blocked by AB or TM. Thus, hepatic disorder is considered a risk factor for aggravation of OHP-induced CIPN in humans and mice, where HMGB1 might play a key role.

Taxanes frequently cause chemotherapy-induced peripheral neuropathy (CIPN). However, the mechanisms underlying CIPN pathogenesis are not fully understood. We previously showed that taxanes preferentially impair Schwann cells (SCs) by inducing dedifferentiation. In this study, we further examined the roles of dedifferentiated SCs in the development of CIPN. We found that mRNA expression of an inflammatory factor, X, was increased in dedifferentiated SC culture or the mouse sciatic nerve after paclitaxel (0.01 μM) treatment or repeated i.p. injection of paclitaxel (20 mg/kg), respectively. Furthermore, murine macrophage cell line (RAW264.7) showed a chemotaxis response toward the conditioned medium of paclitaxel-treated SCs. Consistent with this, we found that the perineural application of an inflammatory factor derived from dedifferentiated SCs induced infiltration of macrophages into the sciatic nerve and mechanical hypersensitivity in mice. Taken together, our findings allow us to conclude that, in response to paclitaxel treatment, an inflammatory factor is released from dedifferentiated SCs to chemoattract macrophages. These SC-dependent macrophage migration may participate in paclitaxel-induced CIPN pathogenesis.

We have previously demonstrated that advanced glycation end product (AGE)-aggregated albumin in the blood of diabetic mice induces acidification and apoptosis in mesangial cells(MCs).Our aim here is to clarify whether it could cause mesangial mitochondrial dysfunction. MCs were incubated with AGE-cholesterol aggregated albumin(ACAA),treated with TMRM(an indicator of mitochondrial membrane potential) or Caspase 3/7 reagent for apoptosis, and then analyzed by FACS.MCs were divided into 3 groups based on size and complexity; P1 small and complex, P2 large and complex, P3 small and simple.MC counts were similar in P1,P2 and P3 of the ACAA and EMEM(control) groups 3 hr after ACAA.Mitochondrial membrane potential(MMP) was 20 % higher in the ACAA group than in the EMEM group.On the other hand,MC counts in P1 were 80 % higher,and in P2 40 % lower,in the ACAA group than in the EMEM group 18 hr after ACAA.MMP in P1 was 45 % higher,and in P2 45 % lower,in the ACAA group than in EMEM group.The number of Caspase 3/7 positive MCs in ACAA group increased in P1,and total Caspase 3/7 fluorescein intensity in P1 was 3 times greater than that in P2 18 hr after ACAA.In the EMEM group,there was no difference in total Caspase 3/7 fluorescein intensity between P1 and P2.Mitochondria could be activated in MCs just after uptake of ACAA,and then gradually inactivated.Decreased MMP could lead to decreased levels of ATP and then MCs apoptosis and necrosis.

[Background] Diabetes is a major risk factor for chronic kidney disease (CKD). However, the mechanisms of diabetes-induced kidney injury remain to be fully elucidated. Here, we focused on old astrocyte specifically induced substance (OASIS), a transcriptional factor, because OASIS mRNA is increased in kidneys of CKD patients in the Nephroseq database. The aim is to determine the pathological roles of OASIS in diabetes-induced kidney injury.

[Methods/Results] C57BL/6J mice were injected with STZ to induce diabetes. Laser microdissection and immunoblotting revealed that OASIS was upregulated in glomeruli of STZ-treated mice. OASIS was detected in podocytes by immunohistochemical staining. To examine the roles of OASIS in podocytes, we generated podocyte-specific OASIS knockout mice (CKO). Mice were subjected to unilateral nephrectomy (UNx) before STZ injection to accelerate kidney injury. After UNx-STZ treatment, the level of serum creatinine (sCr) and the rate of kidney weight to body weight (Kw/Bw) got lower in CKO, compared with control (sCr (mg/dL): control; 0.85±0.11, CKO; 0.59±0.14, Kw/Bw (mg/g): control; 15.0±1.5, CKO;12.4±1.2, P<0.05, N=3-5).

[Conclusion] Podocyte OASIS could be a therapeutic target for the treatment of diabetic kidney disease.

Purpose: We investigated the possible mechanism which central angiotensin II (Ang II) facilitates micturition reflex focusing on the Ang II type 1 receptor (AT1R), GABAR or corticotropin-releasing factor (CRF)R. And, we examined whether a centrally acting AT1R antagonist telmisartan (TEL) ameliorates the central Ang II induced stimulation of micturition reflex. Materials and Methods: Male Wistar rats were anesthetized with urethane, and cystomety was performed. TEL, GABA_AR agonist (muscimol: Mus), GABA_BR agonist (baclofen: Bac) or CRF1R antagonist (CP154526: CP) was icv administered before icv Ang II administration in the rats. Some rats were perorally administered with TEL (10 mg/kg/day) or no centrally acting AT1R antagonist (valsartan: Val, 10 mg/kg) for 8 days. Then, Ang II was icv administered in the rats. Results: TEL, Mus, Bac or CP significantly suppressed Ang II induced shortening of intercontraction interval (ICI). Chronic pre-treatment with TEL but not Val inhibited the Ang II induced shortening of ICI. Conclusion: Central Ang II can facilitate the micturition reflex via modulating the AT1R, GABAR or CRF1R. Blocking of the central AT1R might be a therapeutic target for treatment of detrusor overactivity.

Our recent report showed that hydrogen sulfide (H₂S) is a possible relaxation factor in the rat bladder. Because we have shown bladder dysfunctions develop in spontaneously hypertensive rats (SHRs), we compared effects of NaHS and GYY4137 (H₂S donors) on the bladder contractility and the micturition reflex, and H₂S contents in the bladder between 18-week-old male SHRs and normotensive Wistar rats (Wistars). Effects of NaHS (1×10^-8 to 3×10^-4 M) were evaluated on carbachol (10^-5 M)-induced pre-contracted bladder strips. Under urethane-anesthesia, effects of intravesically instilled GYY4137 (10^-8 to 10^-6 M) on the rat micturition reflex were examined. Tissue H₂S contents were measured by the methylene blue method. NaHS-induced maximal relaxation was significantly higher in the strips of Wistars than those of SHRs. GYY4137 significantly prolonged intercontraction intervals in Wistars, but not in SHRs. The H₂S content in the bladder of SHRs was significantly higher than that in Wistars. These results suggest that H₂S-induced bladder relaxation in SHRs is impaired, which might be a cause of hypertension-mediated development of bladder dysfunctions, thereby resulting in a compensatory increase of the H₂S level in the SHR bladder.

Mitochondrial Ca²⁺ uptake plays an important role for regulating Ca²⁺ signals in vascular smooth muscle cells (SMCs). However, Ca²⁺ affinity of mitochondrial Ca²⁺ uniporter (MCU) is very low. Mitofusin (Mfn) proteins are known to tether endoplasmic reticulum (ER) and mitochondria. Mfn1 is expressed in mitochondria membrane, whereas Mfn2 is localized in mitochondria and ER membranes. In the present study, we examined the physiological roles of Mfn proteins on Ca²⁺ signals in vascular SMCs. SMCs were enzymatically isolated from rat thoracic aorta. The cell proliferation was reduced by siMfn2, but not by siMfn1. The co-localization of mitochondria with sarcoplasmic reticulum (SR) was decreased by siMfn2, but not by siMfn1. In siMfn2-treated cells stimulated by vasopressin (AVP), the peak amplitude of [Ca²⁺]_mito was attenuated, whereas that of [Ca²⁺]_SR was not changed. The half duration of [Ca²⁺]_cyt increase was increased by siMfn2. These results indicate that Mfn2 regulates mitochondrial Ca²⁺ uptake without changing the parameters of Ca²⁺ release from SR. In conclusion, Mfn2 enhances Ca²⁺ signal through tethering SR to mitochondria, which promotes vascular SMC proliferation.

Bombesin receptor subtype 3 (BRS-3) is an orphan G protein–coupled receptor. Based on the obese phenotype of male BRS-3–deficient mice, BRS-3 has been considered an attractive target for obesity treatment. Here, we developed a novel selective BRS-3 agonist (compound-A) and evaluated its antiobesity effects. Compound-A showed anorectic effects and enhanced energy expenditure in dietinduced–obese (DIO)-F344 rats. Moreover, repeated oral administration of compound-A for 7 days resulted in a significant body weight reduction in DIO-F344 rats. To investigate the underlying mechanisms of BRS-3 agonist effects, we focused on the suprachiasmatic nucleus (SCN), the main control center of circadian rhythms in the hypothalamus, also regulating sympathetic nervous system. Compound-A significantly increased the messenger RNA expression of Brs-3, c-fos, and circadian rhythm genes in SCN of DIO-F344 rats. On this basis, energy expenditure enhancement by compound-A may be due to a circadian rhythm change in central and peripheral tissues, enhancement of peripheral lipid metabolism, and stimulation of the sympathetic nervous system. According to these results, BRS-3 agonist might be a good option for obesity treatment with circadian rhythm change and increase of energy expenditure.

Thermogenesis is enhanced not only by cold exposure but also by feeding, which is considered as a partial defense mechanism against obesity. However, the molecular mechanism of diet-induced thermogenesis has remained unclear. Here we found that metallopeptidase nardilysin (NRDC) expression in liver is increased by fasting and decreased by re-feeding in wild-type mice. To elucidate the liver-specific role of NRDC in energy metabolism, we established hepatocyte-specific NRDC deficient mice (LKO). These mice showed intriguing phenotypes including 1) elevation of thermogenic genes in BAT, 2) decrease in lipid accumulation in BAT, and 3) increase in whole-body energy expenditure. These results suggested that the loss of NRDC in hepatocyte enhances adaptive thermogenesis in BAT by an inter-organ metabolic network. Notably, the phenotypic difference between control and LKO was completely eliminated by hepatic vagotomy or elevation of ambient temperature to thermoneutral range (30℃). Furthermore, LKO showed a significant increase in skin blood flow of the plantar at room temperature (23℃), suggesting that heat dissipation is enhanced in LKO. Taken together, these results indicate that hepatic NRDC regulates skin blood flow, thus heat dissipation via nervous system. BAT thermogenesis was then enhanced to compensate for the heat loss. In conclusion, NRDC, a novel sensor of nutrition, mediates diet-induced thermogenesis.

Heparan sulfate (HS) is a highly sulfated glycosaminoglycan distributed on the cell surface and in the extracellular matrix. HS is involved in diverse biological events including embryonic development, angiogenesis and tumor metastasis. Recent studies revealed the pathophysiological involvement of HS in diabetes and we showed HS had a great impact on pancreatic β-cell function. However, the roles of HS in adipose tissue, an important tissue for glucose metabolism, remained to be elucidated.

First, we evaluated the roles HS in 3T3-L1, a cell line of mouse adipocytes. Biochemical assays indicated that HS promoted differentiation of 3T3-L1 possibly via enhancement of FGF signaling. Next, we generated and phenotyped white adipocyte-specific HS-deleted mice (cKO). Several differentiation markers in cKO adipocytes were decreased, emphasizing the important role of HS in adipocyte differentiation. We also confirmed the reduction in gene expression of essential factors for insulin-dependent glucose transport in cKO adipocytes. Indeed, glucose tolerance test revealed glucose intolerance due to insulin resistance in cKO mice. These results demonstrated that HS played an important role in adipocyte differentiation, leading to normal insulin sensitivity and glucose homeostasis.

Background:It is known that body fat mass decreases by exercise under hyperoxia. Besides, hypoxia plays an important role in maintenance of stem cell niche in regenerative medicine field. Although it is obvious that the oxygen partial pressure affects cell proliferation, the relationship between pressure culture condition and cell signal has hardly been elucidated. Control of adipocyte proliferation is an indispensable technology in regenerative medicine to treat obesity and adipose cell transplantation as a fundamental therapy for lipodystrophy. So, in this study, we examined the effect of adipocyte differentiation under the high-pressure cultivation. Methods:3T3-L1 cells were kept under the normal pressure condition or pressurized condition during differentiation for 14 days. Intracellular lipid droplet was evaluated by Oil Red O staining and expressions of adipogenic genes were determined by real time PCR. Results & Conclusions:Lipid droplet and mRNA level of adipogenic genes decreased under the high-pressure cultivation compared to the normal pressure. These data suggest that the high-pressure condition prevented adipocyte differentiation by suppressing expression of adipogenic genes, which induced a decrease in intracellular lipid droplet.

Obesity is associated with proliferation and differentiation of adipose-derived stem cells (ADSCs) into mature adipocytes. Nutritional stimuli induce ADSCs proliferation and differentiation, and this process is well established because master regulators of adipogenic differentiation, C/EBPalpha and PPARgamma were identified. However, under normal condition, molecular mechanisms to maintain stemness of ADSCs are largely unknown.

To identify genes essential for the maintenance, microarray analysis was performed on murine ADSCs and 4-day cultured ADSCs (preadipocytes). Among the 223 up-regulated transcriptional factor genes in ADSCs, we focused on nuclear receptor 4a (Nr4a) family, which play diverse roles including metabolic processes. Nr4a-overexpressed preadipocytes showed reduced accumulation of lipid droplet and decreased expressions of C/EBPalpha and PPARgamma. ChIP analysis confirmed that Nr4a directly bound to C/EBPalpha and PPARgamma promotor. Nr4a family is induced by multiple signals including cyclic AMP in a cell type-specific manner. Application of a cAMP analogue to ADSCs induced Nr4a expressions and decreased expressions of PPARgamma. These data suggested that Nr4a inhibited ADSCs differentiation in a cAMP-dependent manner.

【Introduction】In Parkinson`s disease (PD), α-synuclein (αSyn) accumulation and inclusion triggers dopamine neuronal death and synapse dysfunction in vivo. We previously reported that fatty acid-binding protein 3 (FABP3) is highly expresses in dopaminergic neurons and aggravates αSyn oligomerization when exposure to 1-Methyl-1,2,3,6-tetrahydropiridine (MPTP) in vivo and in vitro. We here discovered FABP3 ligands inhibiting α-synuclein oligomerization induced by arachidonic acid (AA).

【Method 】 FABP ligands were modified from FABP4 inhibitor BMS309403 and assessed their inhibitory action on AA-induced α-synuclein oligomerization using FABP3 and αSyn co-overexpressed Neuron2A cells. αSyn oligomerization levels were measured using western blotting assay and immunohistochemical analyses.

【Summary】AA treatment triggered αSyn oligomerization in Neuro2A cells in FABP3-dependent manner. A potent FABP3 ligand 1 totally blocked αSyn oligomerization and aggregation induced by FAPB3 and AA. In addition, ligands 7 and 8 also elicited inhibition of αSyn oligomerization in Neuro2A cells. Taken together, the new FABP3 ligand is attractive therapeutic candidate for Parkinson and Lewy body diseases.

Progranulin is a multipotent protein that contributes to various pathology such as inflammation and tumorigenesis. Absence of progranulin gene causes the onset of frontotemporal lobar degeneration (FTLD) and neural ceroid lipofuscinosis. Recently, it has been reported that some Parkinson's disease patients with α-synuclein lesions have a progranulin gene mutation. However, the relationship between progranulin and α-synuclein accumulation mechanism is unclear. We examined the effect of progranulin against α-synuclein accumulation.

We evaluated progranulin effects against MPP⁺ damage in human derived neuroblastoma cells (SHSY-5Y cells). A-synuclein and autophagy related factors (AMPK, mTOR, LC-3, p62) were evaluated by Western blot. To clarify autolysosome formation, we used DAL-Green, specific autolysosome detector.

A-synuclein expression was reduced by progranulin treatment. On the other hand, AMPKor mTOR activation were not changed. Furthermore, LC-3Ⅱ/LC3-Ⅰ ratio and p62 expression were reduced. These results indicate that progranulin ameliorated autolysosome formation decreased by MPP⁺.

These findings indicate that progranulin promotes autophagy degradation by inhibiting autolysosome formation and reduces α-synuclein accumulation.

One of the pathological hallmarks of Alzheimer's disease (AD) is senile plaque composed of amyloid-β peptide (Aβ) in the brain. Neprilysin (NEP) is a potent Aβ degrading enzyme. Therefore, identification of the mechanism for NEP activity may lead to development of a preventive approach for AD. Previously we showed that somatostatin (SST) regulates NEP activity. However the molecular mechanism by which SST regulates NEP in the brain is unclear. Recently, We found that cortex/hippocampus-basal ganglia communication is important to upregulate NEP upon SST stimulation. The aim of this study is elucidation of the molecular mechanism of NEP activity based on the cell-cell communication. Firstly proteomics using primary neurons treated with SST identified α-endosulfine (ENSA), an endogenous ligand for a potassium channel, as a novel NEP regulator. To investigate the function of ENSA in vivo, we generated ENSA knock out mice using CRISPR/Cas9 and found that deficiency of ENSA increased NEP activity in the brain. Finally administration of Diazoxide which is a potassium channel modulator decreased amyloid pathology in AD model mice mediated by activation of NEP. Thus, we found a novel preventive approach for AD based on the cell-cell communication.

Amyloidosis causes organ dysfunction due to deposition of β-sheet structured amyloid fibrils in multiple organs. Drugs that break up amyloid fibrils (amyloid breakers) is considered as promising therapy for amyloidosis, but none is yet clinically available. Recently, we performed in vitro fluorescence-based high throughput screening (HTS) (1280 compounds) to identify small molecules that interact with a mutant transthyretin (TTR), a causative protein of hereditary ATTR (ATTRm) amyloidosis. Here, we focused on the HTS-derived nineteen hit compounds and identified two compounds (B and R) that decrease preformed mutant TTR amyloid fibrils. We next validated 113 analogs harboring common basic chemical structure with compound B, and demonstrated that 12 compounds significantly disrupt preformed mutant TTR amyloid fibrils. Importantly, among these 12 compounds, several compounds also broke preformed amyloid fibrils derived from amyloid-β (Aβ) and tau proteins, well-known amyloid causative proteins in Alzheimer's disease. Finally, some of the hit compounds are the component of natural products with low cytotoxicity. Overall, the study will be of great interest for the development of not only therapeutic drugs but also healthy supplements that are applicable for amyloidosis.

The central nervous system has been considered as an organ devoid of lymphatic vessels. Meningeal lymphatic vessels were discovered and it is thought that elucidating mechanism of these vessels contributes to the studies on neurodegenerative diseases such as Alzheimer's disease and multiple sclerosis. Previous researches focused on material flow in the brain and excretion of proteins and immune cells. There are few researches directly investigating the relationship between the brain and meningeal lymphatic vessels. Our research focused on structure of brain cells and neural activities. Meningeal lymphatic vessels connect to the deep cervical lymph nodes (dcLNs). Ligating efferent lymphatic vessels of dcLNs lead to the reduction of cerebrospinal fluid (CSF) drainage. Electrocorticography (ECoG) was recorded for 8 weeks and glial cells and synapses were immunostained in mice 1, 4 and 8 weeks after ligation. Results showed that reducing lymphatic flow concerned neural activities.

[Background and Aim] SIRT1, an NAD⁺-dependent protein deacetylase, exerts cytoprotective effects. We previously reported that resveratrol, an activator of SIRT1, attenuates skeletal muscle pathology in a mouse model of Duchenne muscular dystrophy. The purpose of this study was to elucidate the function of SIRT1 in skeletal muscle using muscle-specific SIRT1 knockout mice (SIRT1MKO).

[Method and Result] Treadmill running distance and inverted net hanging time were significantly shorter in SIRT1MKO than those in wild-type mouse (WT). Blood level of creatine kinase, a marker of muscle membrane injury, after treadmill exercise was significantly higher in SIRT1MKO (2201 ± 407 U/L) than WT (481 ± 99 U/L). Furthermore, Evans blue uptake into muscle cells, which reflects plasma membrane rupture, after treadmill was increased in SIRTMKO than that in WT (1.8% vs. 0.5%, P<0.05). Histological analyses of quadriceps muscle showed that central nuclei, an indicator of muscle regeneration, and atrophied muscle fibers with (<1,500 μm²) were significantly increased in SIRT1MKO compared to those in WT.

[Conclusion] These results suggest that SIRT1 maintains muscle function and attenuates muscle membrane injury.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disease characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor gain-of-function mutations in ACVR1 (also known as ALK2), a type I receptor for bone morphogenetic protein (BMP). Despite numerous studies, no drugs have been approved for FOP. Here, we developed a high-throughput screening (HTS) system focused on the constitutive activation of FOP-ACVR1 by utilizing a chondrogenic ATDC5 cell line that stably expresses FOP-ACVR1. After HTS 5,000 small molecule compounds, we identified two hit compounds that are effective at suppressing the enhanced chondrogenesis of FOP patient-derived iPSCs (FOP-iPSCs) and suppressed the HO of multiple model mice, including FOP-ACVR1 transgenic mice and HO model mice utilizing FOP-iPSCs. Furthermore, we revealed that one of the hit compounds is a mTOR signaling modulator that indirectly inhibits mTOR signaling. Our results demonstrate that these hit compounds could contribute to future drug repositioning and the mechanistic analysis of mTOR signaling.

Theorientation of actin filaments regulates protrusion and contractile movement of cells because it determines the direction of actin elongation and myosin movement. Little is known about the orientation change of a single actin filament in living cells because of the lack of the method to probe the orientation of actin filaments in living cells. Here we report a method to visualize the orientation of single actin filament. We labeled N and C-termini of tropomyosin with different fluorescent dyes, and introduced it into living cells by electroporation. We performed dual fluorophore-single molecule localization analysis and orientation of actin filaments was determined by the relative position of N and C-termini. We observed the orientation of actin filaments, which flow near focal adhesions(FAs), and found that not only actin filaments rotate rapidly near the FAs, but also they rotate inwardlyin the frontal region of FAs. Actin filaments are also disrupted in the anterior region of FAs. The actin remodeling zone surrounding FAs may be the place for reconstructing the FAs-linked actin network.

The plasma membrane is the site of intercellular communication and subsequent intracellular signal transduction. The specific visualization of the plasma membrane in living cells, however, is difficult using fluorescence-based techniques owing to the high background signals from intracellular organelles. In this study, we show that second harmonic generation (SHG) is a high-resolution plasma membrane-selective imaging technique that enables multifaceted investigations of the plasma membrane. In contrast to fluorescence imaging, SHG specifically visualizes the plasma membrane at locations that are not attached to artificial substrates and allows high-resolution imaging because of its subresolution nature. These properties were exploited to measure the distances from the plasma membrane to subcortical actin and tubulin fibers, revealing the precise cytoskeletal organization beneath the plasma membrane. Thus, SHG imaging enables the specific visualization of phenomena in the plasma membrane with unprecedented precision and versatility and should facilitate cell biology research focused on the plasma membrane.

Oral administration is widely used in pharmacological experiments. However, the most common method, oral gavage, causes stress responses through restraint and insertion of a needle into the stomach, often affecting subsequent animal behaviors such as sleep/wake. Here, we developed a new method of voluntary oral administration: PERO. In PERO, we used a mixture of viscous paste foods (e.g. sweetened condensed milk) and a solvent (dimethyl sulfoxide) as vehicle. Mice rapidly and completely consumed the vehicle by licking, and the behavior persisted for 4 weeks when repeated daily. Plasma corticosterone levels showed that our method was significantly less stressful than oral gavage. Moreover, we demonstrated the utility of the method through an assay for sleep-inducing effects of a water-insoluble orexin antagonist. In conclusion, PERO will be useful especially in behavioral neuroscience vulnerable to stress responses, and will improve animal welfare.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive disease of unknown cause. Under the pathogenic environment, myofibroblasts (MyoFs) mainly differentiated from fibroblasts play a key role in lung fibrogenesis. Established MyoF has been considered as an irreversible phenotype, but recently shown to dedifferentiate to fibroblast. This feature is desirable in development of pharmacologic strategy against IPF because most patients with IPF appear to accumulate pathogenic MyoFs in their lungs at the time of clinical presentation. Therefore, we have established several strains of primary cultured MyoFs from the fibrotic lungs of patients. Using our MyoF assay system, the drug library for compounds that inhibit epigenetics-related signals was screened by monitoring downregulation in expression of collagen and MyoF markers, α-SMA and ED-A fibronectin. Through this assay, we found in vitro that a certain histone methyltransferase inhibitor potently dedifferentiated MyoFs. In addition, intratracheal administration with the compound at the early fibrotic stage of bleomycin-injured lung successfully ameliorated lung fibrosis in mice. We will discuss the mechanism by which the compound affects pathogenic myofibroblast under lung fibrogenesis.

Mucus hyperproduction is a hallmark of chronic airway inflammatory diseases, such as asthma and COPD. Mucus production in a population of patients with severe asthma does not respond to steroids. However, the underlying mechanism of this steroid-resistance remains incompletely understood. In our pharmacological study with house dust mite (HDM)-sensitized asthmatic mice, we have found that MUC5AC mucus gene expression was steroid resistant, although the number of eosinophil was significantly decreased. In these mice treated with DEX, the expression level of IL-17A and IL-13 was considerably high, as well as MUC5AC. We also found that intratracheal administration of both IL-17A and IL-13 was sufficient to induce steroid-resistant MUC5AC production in lung, whereas IL-17A or IL-13 alone was not. Furthermore, HDM-induced steroid-resistant MUC5AC production was partially reversible with DEX in mice lacking IL-17A. Collectively, these results suggest that the coordinated action of IL-17A and IL-13 mediates steroid-resistant mucus production in mouse model of asthma.

Background RhoA/Rho kinase and PKC/CPI17mediated signaling can inhibit myosin phosphatase to induce contraction in smooth muscles. However it is still unclear how the signaling plays an important role to regulate gastrointestinal motility in vivo.

Aim Intestinal and colonic contractile and transit abilities were tested using CPI17 deficient (KO),phospho-inactive mutant CPI17knock in (TA) and wild type mice (WT).

Methods Isometric force and phosphorylation of Myosin light chain (MLC) and CPI17 stimulatedwith Carbochol(Cch)were measured. Transit abilities were observed by FITC dextran and dextran　beads method, respectively.

Results High concentration of KCl induced contractions were no difference among WT, KO and TA of ileal and colonic circular muscles. However the sustained contraction by Cch of ileum and colon in KO and TA were decreased compared with WT. The MLC phosphorylation level was also lower in KO and TA than WT. Ileal transit ability did not change in KO and TA compared with WT.In contrast,colonic transit in KO and TA were significantly delayed compared with WT.

Conclusion CPI17 is important to maintain the sustained contraction of gastrointestinal tract due to receptor stimulation in vitro. In addition, the PKC/CPI17 pathway is more important for maintenance in colon transit ability than ileal part in vivo.

Background: Cirrhosis is a condition come from excessive liver fibrosis and followed by serious second diseases. 1.3 million people are died of cirrhosis in a year but there is no effective therapeutic medicine. α7 nicotinic acetylcholine receptor (α7nAChR), initially found a receptor related to neurotransmission, expresses on immune cells and activation of this receptor leads anti-inflammatory effect. However, there is few reports showing the relationship between α7nAChR and fibrosis.

Aim: Using α7nAChR knocked out mice (α7 KO), we investigated whether α7nAChR has any effect on liver fibrosis.

Methods: Liver fibrosis model mice were established with carbon tetrachloride (CCl₄, 1 ml/kg, twice a week). The amount of collagen and pro-fibrotic mRNA expressions in livers were measured at 1.5 and 4 weeks.

Results: α7 KO treated with CCl₄ showed significant decrease in liver fibrosis at 4 weeks compared to wild type mice (WT). Furthermore, mRNA expressions of Acta2, TGF-β1 and Col1a1 in α7 KO were significantly lower than WT at 1.5 weeks.

Conclusion: Increase of pro-fibrotic mRNA expression and liver fibrosis induced by CCl₄ were alleviated in α7nAChR KO mice. These data suggested that α7nAChR might be a therapeutic target for cirrhosis.

N-acetyl-p-aminophenol (APAP), Concanavalin A (ConA) and Carbon tetrachloride (CCl₄) exposure generated drug induced liver injury models. Overdose of APAP is the most common cause of acute liver failure, as it induced mitochondrial oxidative stress and hepatic necroptosis. Also, ConA and CCl₄ are causing immune-mediated liver injury and centrilobular necrosis, respectively. We conducted this study to evaluate the effects of Mito-TEMPO (Mito-T) on acute liver injury models in mice. An injection of Mito-T (20 mg/kg) after 1h of APAP (400 mg/kg) administration markedly inhibited the elevation of serum transaminase activity but was not able to attenuate the ConA (12.5 mg/kg) and CCl₄ (0.025 mL/kg) hepatotoxicity in C57BL/6J mice. Mito-T significantly reduced the parameters of APAP derived oxidative stress, such as mitochondrial radical production and nitrotyrosine formation in mouse liver; though the decreased glutathione (GSH) level and c-Jun N-terminal Kinases (JNK) activation were not suppressed in liver. In addition, Mito-T was hepatoprotective after 4 hours of APAP ingestion, whereas, N-acetyl-L-cysteine, the only recognized therapeutic agent against APAP hepatotoxicity, became less effective with the time lapse.

We demonstrated that Mito-T alleviates mitochondrial oxidative stress and suppresses the APAP-induced liver injury in mice. The results suggest that Mito-T could be a promising novel therapeutic agent for APAP overdosed liver injury.

Astrocytes are crucial for synaptic plasticity and memory formation. Astrocyte-derived factors, such as D-serine and lactate, have been suggested to enhance learning and memory likely via modulating NMDA receptor functions in neurons. However, the intracellular mechanisms of astrocytes that release these factors are not fully clarified. Here we adopted an optogenetic approach to regulate the intracellular cyclic AMP (cAMP), a major second messenger, specifically in astrocytes in vivo. We developed a transgenic mouse line in which astrocytes express photoactivated adenylyl cyclase (PAC), a protein that rapidly changes its conformation and synthesizes cAMP from ATP in response to blue light. In these mice, we optogenecically modulated the intracellular levels of cAMP in hippocampal astrocytes and investigated the effect of a prolonged cAMP elevation on spatial memory. We found that a long-term cAMP elevation in astrocytes after training session facilitated the memory fading, whereas a short-term cAMP elevation enhanced memory formation. We also found that astrocytic cAMP facilitates synaptic plasticity through activating NMDA receptors, which may underlie both memory fading and enhanced memory formation. Thus, our results suggest that astrocytic cAMP signaling modulates hippocampus-dependent learning and memory.