Contraction of cardiac muscle is caused by periodic sliding of an array of thin actin filaments into a lattice of thick myosin filaments in the sarcomere, the contractile unit of striated muscles. In actively contracting cardiomyocytes, thin filaments exhibit continuous exchange of actin subunits at their ends, although underlying mechanisms are not well understood. Fhod3, a cardiac member of the formin family proteins, is a likely candidate for a key regulator of actin dynamics in cardiomyocytes. We have shown that Fhod3 is required for cardiac development and the maintenance of the normal contractile function of the heart. Here we show a direct molecular link between Fhod3 and cMyBP-C, a thick myosin filament-associated protein that modulates myocardial contraction via cross-bridge arrangement. The direct interaction between Fhod3 and cMyBP-C appears to serve to control the Fhod3-mediated actin turnover in a manner that depends on cross-bridge arrangement. Indeed, overexpression of Fhod3 in the absence of cMyBP-C adversely affected cardiac function with a defect of sarcomere integrity. We will discuss the underlying mechanism and also discuss the possibility that targeted disruption of this cross-talk machinery leads to an artificial modulation of cardiac contractility.

G-protein activated inwardly rectifying potassium (GIRK, Kir3) channel is one of the effectors in signal pathways from ethanol, opioid, dopamine, and other addictive substances. We found associations between genetic polymorphisms in the GIRK subunit genes and sensitivity to addictive substances in mice and humans. We found that fluoxetine and paroxetine, selective serotonin reuptake inhibitors (SSRIs), but not fluvoxamine, another SSRI, inhibited GIRK channels in vitro and reduced preference for methamphetamine in mice. In addition, we found that ifenprodil, a widely used drug for dizziness, also inhibited GIRK channels in vitro. Another research group has shown that ifenprodil reduced preference for addictive substances using rodents. Furthermore, we found that relapse rate and relapse risk scores were lower in alcoholics who were treated with GIRK inhibitors. We also demonstrated an inhibitory effect of ifenprodil on alcohol use in patients with alcohol dependence in a prospective, randomized, controlled, rater-blinded study. These results suggest that GIRK channels are important molecules in the reward system and candidate targets for pharmacotherapy of drug dependence.

We found and study new three molecules related methamphetamine dependence, Shati/Nat8L, Piccolo and TMEM168. Accumbal Shati rescue the dependence-induced by methamphetamine via mGluR3, and dopaminergic deficiency. Reduce of Piccolo can inhibit methamphetamine dependence via GABA regulation. TMEM168 also inhibit the methamphetamine dependence via osteopontin pathways. We found some pathway to establish drug dependence. We expected them for the new medical target for the drug dependence.

Recently, we attempt to find these molecular regulation system to develop new medical tool. Here we would like to recent our results

Stress potentiates rewarding properties of cocaine. To elucidate neural mechanisms underlying this effect of stress, we developed an experimental paradigm combining cocaine-induced conditioned place preference (CPP) with a restraint stress. Acute restraint stress exposure immediately before posttest significantly increased cocaine CPP scores. It has been suggested that the extracellular noradrenaline (NA) level is increased by stress in the laterodorsal tegmental nucleus (LDT), which sends cholinergic projections to dopamine (DA) neurons in the ventral tegmental area (VTA), and in the medial prefrontal cortex (mPFC), which receives DA input from the VTA. Thus, we investigated the roles of NA in these brain regions. Intra-LDT injection of an α2 adrenoceptor antagonist or intra-mPFC injection of an α1 adrenoceptor antagonist attenuated the stress-induced enhancement of cocaine CPP. In vitro whole-cell recordings revealed that α2 adrenoceptor stimulation reduced GABAergic inputs to LDT cholinergic neurons and that α1 adrenoceptor stimulation directly excited mPFC pyramidal neurons. These findings suggest that stress-induced increases in neuronal activity of the LDT and mPFC contribute to the enhancement of rewarding properties of cocaine.

Nalmefene, an opioid system modulator, is approved in the EU and other countries for as-needed use to reduce alcohol consumption in patients with alcohol dependence. Accumulating evidence indicates that the endogenous opioid system has important roles in alcohol dependence. Alcohol stimulates the release of endogenous opioid peptides such as β-endorphin and dynorphin in the brain. β-endorphin activates μ-opioid receptor leading to euphoric mood and positive reinforcement, while dynorphin activates κ-opioid receptor leading to dysphoric mood and negative reinforcement. These euphoric/dysphoric mood and reinforcement effects via endogenous opioid systems are suggested to be implicated in repeated alcohol intake in patients with alcohol dependence.

Nalmefene acts as an antagonist at μ- and δ-opioid receptor and a partial agonist at κ-opioid receptor. Preclinical studies have shown that nalmefene reduced the alcohol intake in alcohol preference rats. In clinical trials, as-needed use of nalmefene with psychosocial support reduced the number of heavy-drinking days and total alcohol consumption. These results suggest that nalmefene modulates the alcohol-induced euphoric/dysphoric mood via opioid system and thereby contribute to reduction in alcohol consumption in patients with alcohol dependence.

 In this symposium, we will discuss the implications of opioid system in alcohol dependence and pharmacological profiles of nalmefene in preclinical and clinical studies.

Non-motile primary cilia are sensory organelles that present in most vertebrate cell types. Its localization within tissue architecture and a growing list of cilia-localized receptors, in particular a limited set of G-protein-coupled receptors (GPCRs), determine a host of crucial physiologies, which are disrupted in human ciliopathies. Melanin-concentrating hormone (MCH) is a cyclic neuropeptide exerting its action through two GPCRs, MCHR1 and MCHR2. The extensive progress using genetic and pharmacological approaches has confirmed that the MCH-MCHR1 system is involved in feeding and possibly emotional processing. We recently found the that MCH signaling through a Gi/o-Akt pathway induces cilia length shortening in ciliary MCHR1-expressing RPE1 cells without no cell cycle progression. This is the first example of effective-neuropeptide-induced cilia length reduction. Here, we discuss our recent progress in the characterization of signaling components that cause cilia shortening via ciliary MCHR1 localized in neuron. Short cilia phenotypes have been associated with various metabolic disorders. Thus, our study has highlighted the unique signaling environment of the primary cilium, in which the design allows for organized signaling in neuronal network toward feeding.

Cilia are microtubule-based appendages that project from the surfaces of various eukaryotic cells and play critical roles in sensing extracellular stimuli and transducing developmental signals. Defects in the assembly and functions of cilia result in a variety of congenital disorders, which are collectively called the ciliopathies.

The bidirectional trafficking of ciliary proteins along the microtubule-based axoneme is mediated by the intraflagellar transport (IFT) machinery, which contains the two large multisubunit complexes IFT-A and IFT-B. Anterograde protein trafficking from the base to the tip of cilia is mediated by the IFT-B complex driven by kinesin-2 motor proteins, whereas retrograde trafficking is mediated by the IFT-A complex driven by the dynein-2 complex.

I will introduce the architecture and function of IFT machinery revealed by utilizing the visible immunoprecipitation (VIP) assay, which we recently developed as a simple and flexible strategy for visually detecting protein-protein interactions, and CRISPR/Cas9 genome editing.

Primary cilia are hair-like sensory organelles whose dimensions and location vary with cell type and culture condition. Herein, we employed scanning ion conductance microscopy (SICM) to visualize the topography of primary cilia from different cell types. By combining SICM with fluorescence imaging, we successfully distinguished between surface cilia that project outward from the cell surface and subsurface cilia that are trapped below it. The nanoscale structure of the ciliary pocket, which cannot be easily identified using a confocal fluorescence microscope, was observed in SICM images. Furthermore, we developed a topographic reconstruction method using current-distance profiles to evaluate the relationship between set point and topographic image and found that a low set point is important for detecting the true topography of a primary cilium using hopping mode SICM.

Primary cilium is a nonmotile sensory organelle that possesses selective membrane receptors. The cilium is dynamically regulated in a cell cycle-dependent manner; it is displayed at the G₀/G₁ phase of many cell types, including neural progenitor cells, and resorbed prior to the S phase. The ciliary dynamics has pivotal roles in development of many tissues/organs. However, the molecular mechanism how the cilium controls cell proliferation remains largely unknown. We found that IGF-1 was one of the growth factors that promoted proliferation of neural progenitors. Tctex-1, a light chain of cytoplasmic dynein that plays a key role in ciliary resorption, can be free from dynein complex when it is phosphorylated at Thr 94. We also found that phospho-Tctex-1 was enriched at the ciliary base in the cells. Molecular approaches analyzed in an immortalized retinal pigment epithelial cell line revealed the physical and functional interaction of phospho-Tctex-1 with the regulators of actin dynamics such as annexin A2, Arp2/3 complex and Cdc42, which promoted branched actin polymerization and dynamin- and clathrin-dependent endocytosis at peri-ciliary region. Our study demonstrated that these mechanisms collectively regulate ciliary resorption and proliferation of neural progenitors.

Intestinal commensal bacteria is now recognized to be an important element in the control of the development and function of the host immune system, including the production of secretory IgA and differentiation of specific T cell populations. Although many studies mainly focused on the commensal bacteria in the intestinal lumen or mucus layers, genome-based bacterial analysis using intestinal tissue allowed us to identify Alcaligenes as symbiotic resident bacteria of Peyer's patches (PPs), a major gut-associated lymphoid tissue in the small intestine, which is regulated by type 3 innate lymphoid cells (ILC3) . Our subsequent study showed that Alcaligenes have a greater ability to survive in dendritic cells (DCs) and modulate the production of cytokines such as IL-1b, IL-6, IL-10, IL-12p40, and IL23 from DCs. We recently found that lipopolysaccharide (LPS) of Alcaligenes acts as a weak TLR4 agonist and thus creates a homeostatic inflammatory condition that includes IgA responses in PPs without the excessive pathological inflammation These findings allowed us to apply Alcaligenes LPS could be used as a safe and effective vaccine adjuvant.

Cells respond to external stimuli and eventually make the decision for survival, growth and differentiation. In this process, outcomes of the kinase activities of signal transduction pathways and transcription factors often show dynamically rich, highly quantitative behaviors over time. This quantitative response is, however, eventually converted into a qualitative response and a binary decision (eg. survival or cell death), at the stage of cell decision. A binary decision of a cell is made based on the combined activities of multiple molecules. However, the molecular mechanism of threshold setting of the responses in each cellular context is still unknown. To reveal this mechanism, we analyze the experimental data of signaling and multi-Omics using a mathematical model and extract logical rules in the cell decision mechanism and the target molecules (eg. marker molecules) that define the cellular threshold. 

Our analysis, using NF-kB and ErbB receptor signaling as model systems, indicates that the high-order inter-molecular formation in signaling pathways and epigenetic regulation plays an important role for binary activation of gene expression, and this mechanism might act as a threshold setting for cell regulation. I will introduce our modeling approach for NF-kB signaling pathway, single cell transcriptome and epigenetics.

Sepsis, a syndrome that occurs when microbial invasion induces systemic illness, is one of the most common reasons for critical ill patients to be admitted to an intensive care unit and, despite advances in overall medical care, it represents a major clinical problem and remains the leading cause of death in the critically ill patient population. A major hurdle in the clinical management of patients suffering from the sepsis syndrome is the lack of the effective treatment. Thus, the important goal in critical care medicine is to find out significant therapeutic strategies that would impact favorably on patient outcome. Gene therapy can be considered as one of the most promising novel therapeutic approaches for nasty disorders. Since sepsis can be characterized by the induction of multiple genes and their products, sepsis may be regarded as a gene-related disorder. A number of transcription factors, including NF-kB, AP-1 and STAT3, are profoundly activated during sepsis, and may play a pivotal role in the pathophysiology of sepsis. The decoy strategy has been developed as a useful tool for the involvement of those transcription factors in disease pathology. The decoy oligodeoxynucleotides (ODNs) can specifically compete for the binding elements of those transcription factors, thereby blocking their actions. We have devised the potential usefulness of different transcription factor ODNs for gene therapy of sepsis. Our results suggest that the development of decoy ODN techniques for those transcription factors may provide new perspectives on revolutionary gene-therapy approaches for the fight against sepsis.

Influenza viruses cause worldwide epidemics and pandemics, and sometimes trigger critical illness especially among humans with high risk factors, including obesity, diabetes, and cancers, of which population is increasing with the aging of society. To date no effective preemptive medicine or treatment of severe influenza has been established. Influenza virus is a single stranded RNA virus, and transcription and replication of the virus genome occur in the nucleus. Since viral infection is generally associated with virus-driven hijack of the host cellular machineries, influenza virus may utilize and/or affect nuclear system. Recent high-resolution chromatin interaction maps using chromosome conformation capture (3C) techniques such as 4C and Hi-C have defined units of chromatin that are 3D, termed topologically associated domains (TADs). In the present study, using 4C-seq and ChIP-seq we examined how host chromatin 3D structure and epigenetic modification were changed to influenza virus infection, and how they were involved in the pathology of severe influenza. Our data suggest that host chromatin 3D dynamics could be a novel target of prevention and/or treatment for severe influenza virus infection.

High mobility group box 1 (HMGB1), a damage-associated molecular pattern (DAMP) protein, is considered to play a role in chemotherapy-induced peripheral neuropathy (CIPN), since an anti-HMGB1-neutralizing antibody prevents CIPN in rodents. Thrombomodulin alfa (TMa), a recombinant human soluble thrombomodulin, is capable of promoting thrombin-dependent degradation of HMGB1. TMa inhibits intraplantar HMGB1-induced allodynia and prevents CIPN caused by distinct chemotherapeutics in rodents. Inhibitors of thrombin, vitamin K or factor Xa attenuate or abolish the preventive effect of TMa on oxaliplatin-induced peripheral neuropathy (OIPN). Repeated administration of those agents aggravates the OIPN and elevates plasma HMGB1 levels. Our retrospective cohort study shows that hepatic dysfunction, possibly due to oxaliplatin therapy, is a risk factor for severe OIPN. Our preclinical study demonstrates that hepatic damage promotes OIPN most probably by promoting HMGB1 release in mice. Together, our data suggest that TMa is available for prevention of CIPN, which requires special attention on co-administration of anti-coagulants, and that careful monitoring of hepatic function is useful to predict severe OIPN.

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent side effect of taxanes. Because of the as yet poor understanding of the mechanism underlying CIPN pathogenesis, there is no indicator for objective diagnosis like a biomarker. In addition, treatment options for CIPN remain largely unsatisfactory. Previous our study demonstrated that paclitaxel preferentially impair myelin-forming Schwann cells, and consequently induce dedifferentiation and demyelination of Schwann cells. Recently, in a paclitaxel CIPN mouse model, we found that an inflammatory factor is released from dedifferentiated Schwann cells in the mouse sciatic nerve into the blood, highly correlated with the on-set of pain hypersensitivity. In this presentation, I will introduce the usefulness of this inflammatory factor as a biomarker for the progression of CIPN.

On the other hand, considering our previous findings, it seems that some drugs, which induce differentiation of Schwann cells and supply newly formed mature Schwann cells at sites of demyelinated lesions, may be a new therapy for CIPN. Now, I am promoting therapeutic drug screening for CIPN based on this concept, and I will talk about a part of our results in this presentation.

Chemotherapy-induced peripheral neuropathy (CIPN) is a frequent and disabling side effect of cancer treatment. We evaluated the preventive effects of cryotherapy in a self-controlled trial.

Forty breast cancer patients who were planned to undergo weekly paclitaxel treatments (80 mg/m² for 1 hour) with a cumulative dose of at least 960 mg/m² were enrolled. Each patient wore frozen gloves and socks on the dominant hand and foot, and the non-dominant side acted as the untreated control. CIPN symptoms were assessed by tactile sensitivity, thermal sensitivity, performance speed, and Patient Neuropathy Questionnaire. We defined tactile sensitivity, patients-blinded test, as the primary outcome. We concluded that cryotherapy is useful for preventing both the objective and subjective symptoms of CIPN and resultant dysfunction.

There were other reports about cold intolerance or frostbites due to cryotherapy. In our trial, nonwoven fabric covers were applied to alleviate the discomfort if the patients complained of cold pain. The subgroup analysis indicated that this intervention did not interfere with the effects of the cryotherapy. Further discussion to establish standard cryotherapy with safe and adaptable settings is needed.

Alzheimer's disease (AD) is the most common type of neurodegenerative disorder in the world. Although both amyloid beta peptide deposition and neurofibrillary tangle (NFT) formation in the AD brain have been established as pathological hallmarks of the disease, many other factors contribute in a complex manner to the pathogenesis of AD. Longitudinal pathophysiological processes cause patients' brains to exist in a state of chronic neuroinflammation; additionally, reactive glial cells contribute to AD pathogenesis. However, the detailed molecular and cellular mechanisms underlying this pathogenesis are still unclear. Such disease complexities make it difficult for the pathogenesis of AD to be understood, and impede the development of effective therapeutic strategies to combat the disease. Relevant AD animal models are thus likely to serve as a key resource to overcome many of these issues. In this symposium, I will introduce current situation of research and share perspectives for understanding glial pathophysiology.

Not only neurological disease but also physiological aging affects brain function. Takahashi et al (2016) found that histological and motor functional alterations were associated with age-related decline of brain mitochondrial function. Aged mice (15-month-old) displayed significant reductions in mitochondrial oxygen consumption rate, coenzyme Q (CoQ) content, vesicular glutamate transporter 1 level in the motor cortex, and motor function compared to young mice (6-month-old). CoQ is a coenzyme, and present in the mitochondria. It is an electron transporter in the respiratory chain involved in ATP production. However, age-related electrophysiological impairments of the motor cortex were poorly understood. In this talk, I will describe how physiological aging affects electrophysiological activities in the motor cortex of mice, and present data to show how exogenous CoQ treatment affects the age-related alteration in the aged mice. I will also discuss the influence of exogenous CoQ treatment on the age-related decline of electrophysiological activities in the motor cortex. Takahashi and our studies suggested that the age-related alterations were ameliorated by exogenous CoQ treatment. Although the relation between central nervous system and age-related motor decline remain to be elucidated, our results may serve as the basis for developing therapy of age-related motor impairment.

Memantine ameliorates progressive symptomes in Alzheimer's disease (AD) through moderate inhibition of N-methyl-D-aspartate receptors (NMDARs). Here we report that a novel target of mementine, ATP-sensitive K⁺ (K_ATP) channels are implicated in memory improvement. K_ATP channels Kir6.1 or Kir6.2 are composed with sulfonylurea receptors (SURs), which are distributed both in peripheral tissues and central nervous system. We confirmed that memantine improves both memory impairment and perturbed NMDAR-dependent LTP in APP23 mouse hippocampus. Unexpectedly, memantine in vivo increased CaMKII activity in APP23 hippocampus, and memantine-induced enhancement of hippocampal LTP and CaMKII activity was in vitro abolished by treatment with pinacidil, a specific opener of K_ATP channels. We therefore confirmed that memantine inhibits K_ATP channels Kir6.1 and Kir6.2 and elevates intracellular Ca²⁺ concentrations by inhibition of Kir6.1 or Kir6.2. Kir6.2 was preferentially expressed in the postsynaptic regions, whereas Kir6.1 was predominant in mouse hippocampal neuron dendrites. Finally, we confirmed that Kir6.2 heterozygous mutant mice exhibit severe memory deficits and hippocampal LTP impairment that could not be rescued by memantine administration. Taken together, we propose a novel strategy that memantine inhibits Kirs 6.2/6.1 activities, thereby improving memory impairment in AD patients.

Reactive oxygen species (ROS) is considered as one of the main factors inducing physiological aging. However, molecular mechanisms how ROS induce physiological and pathological aging have not been fully understood.

In the cerebellar parallel fiber (PF)-to-Purkinje cell (PC) synapse (PF synapse), nitric oxide (NO)-dependent long-term potentiation (PF-LTP) is characterized. Previous studies indicated that the PF-LTP is dependent on S-nitrosylation of type I ryanodine receptor (RyR1), an intracellular Ca²⁺-release channel, and the resulting novel type of Ca²⁺-release, nitric oxide-induced Ca²⁺-release (NICR) in cerebellar PCs.

Thiol groups in cysteine residue are the target of S-nitrosylation of proteins by NO as well as the target of disulfidation (disulfide-bond formation) by ROS. Thus, it is highly possible that protein disulfidation blocks the induction of S-nitrosylation and the resulting S-nitrosylation-dependent biological events, such as PF-LTP.

In this symposium, I will introduce our recent studies designated to test this hypothesis. In the cerebellar slices pretreated with ROS, S-nitrosylation of RyR1, NICR and PF-LTP were impaired. Furthermore, in the cerebellar slices from aged mice (about 2-years old), RyR1 S-nitrosylation, NICR and PF-LTP were again inhibited. These results support the hypothesis, and also suggest that endogenous ROS possibly induce physiological aging through the inhibition of S-nitrosylation in old animals.

Hypoxia is a condition in which the tissue is deprived of adequate oxygen supply. It occurs in several cardiovascular disorders, such as cardiac hypertrophy and myocardial ischemia, and accelerates the inflammatory processes. While each cell exerts its own responses to hypoxia, most of them are mainly mediated through the transcription factor, hypoxia inducible factor-1a (HIF-1a) and HIF-2a. Macrophages are key mediators of inflammation, and can be broadly classified as M1 (pro-inflammatory) and M2 (anti-inflammatory) type. We found that HIF-1a and HIF-2a is specifically expressed in M1 and M2 macrophages respectively. The balance between HIF-1a and HIF-2a, termed as HIF-a switching, regulates macrophage activation and its resolution. We further examined the roles of macrophage hypoxia signaling in cardiac remodeling using mice transverse aortic constriction model, and found that M1 macrophages accumulate into the hypoxic area though a HIF-1a dependent manner. As an underlying molecular mechanism, we discovered that HIF-1a mediated glycolytic reprograming is critically required in macrophage migration potential. Importantly, LV hypertrophy and cardiac fibrosis were more prominent while systolic function was impaired in HIF-1a knockout mice. These results demonstrate a novel functional link between hypoxia activated cardiac macrophage and cardiovascular remodeling.

The blood vessel is important tissue structures to deliver oxygen, nutrition and so on. An abnormal blood vessel formation is a common feature of tumor tissue that were characterized by hyper-permeability, irregular vascularization, immature vessels and intravasation. Therefore, tumor tissue is exposed to low oxygen nutrition depletion and low pH due to hypoperfusion and elevated interstitial pressure. These environments are one of the reasons for chemo- and radio-resistance. Previously, we reported that prolyl hydroxylase (PHD) inhibitor induced tumor blood vessel normalization and improved tumor microenvironment in tumor mouse model. However, effects of PHD inhibitor on tumor progression is controversial. Enhanced hypoxia inducible factors (HIFs) signaling in cancer cells act to promote cancer proliferation and metastases. On the other hand, increasing of HIFs signaling in immune cells may lead to activate inflammation and elicit anti-tumor effect. In this session, we will talk about our study how PHD inhibitor improved tumor microenvironment and focused on tumor infiltrate immune cells were phenotypic alteration after PHD inhibitor treatment in mouse model. We will also discuss about usefulness of PHD inhibitor for anti-cancer therapy.

Loss of prolyl hydroxylase 2 (PHD2) activates hypoxia-inducible factor (HIF)-dependent hypoxic response including enhanced anaerobic glycolysis, which releases great amount of lactate from cells into circulation. However, surprisingly, systemic activation of hypoxic response by PHD2 inhibition in mice did not lead to hyperlactatemia. This serendipitous phenomenon led us to hypothesize that the activated hypoxic response enhances Cori cycle, the lactate-glucose carbon recycling system between muscle and liver, and then reduces circulating lactate level. Here we show that liver-specific inactivation of PHD2 improves the survival of lactic acidosis by activating Cori cycle in the liver, and pharmaceutical inhibition of PHDs also improves the survival of lethal lactic acidosis induced by endotoxin shock. Lactic acidosis is also known to be induced by metformin, which is a popular therapeutic for type 2 diabetes mellitus and also has anti-cancer and anti-aging properties but is contraindicated in individuals with chronic kidney disease (CKD) due to the risk of metformin-associated lactic acidosis (MALA). We also report that treatment with a PHD-inhibitor per os significantly improves the survival rate of MALA in CKD mice. Our findings would provide a new concept that the oxygen sensor PHDs serve as new therapeutic targets for the treatment of endotoxin shock-induced lactic acidosis or MALA. The application of PHD-inhibitor as the rescue agent for the renal anemia or myocardial infarction will be also discussed.

In chronic kidney disease (CKD), tubulointerstitial hypoxia is regarded as a final common pathway leading to end-stage kidney disease. Insufficient oxygenation negatively influences the balance between injury and repair in tubular epithelial cells.

Studies on erythropoietin (EPO) transcription led to the identification of hypoxia inducible factors (HIFs) and their key regulators, prolyl hydroxylases (PHDs). Based on these, several small molecule PHD inhibitors are developed for the treatment of anemia in CKD, which are currently in phase II/III clinical trials. Studies so far demonstrate successful increases in hemoglobin levels by raising plasma EPO levels and optimizing iron utilization.

Application of PHD inhibitors has several potential implications beyond anemia treatment, and there is a promising view that activation of the HIF signaling might protect the ischemic kidney from injury. This concept is extensively tested in multiple acute kidney injury models, but knowledge is limited in the context of CKD. Some studies demonstrate the protective effects of ameliorating inflammation and reducing oxidative stress. In human clinical studies, some of the PHD inhibitors exhibit the additional advantage in terms of glucose and lipid metabolism, which may be beneficial for the treatment of metabolic kidney disorders. On the other hand, negative consequences of sustained HIF activation are also reported, including renal fibrosis and aggravation of polycystic kidney disease. Renal consequences are likely determined by multiple systemic effects of PHD inhibition and may thus differ depending on the clinical context and the pathological stages.

Various physiological and pathological processes are accompanied with the alteration of extracellular local pH. Accordingly, there has been a strong demands for the development of methods to analyze the cell surface pH. We established a novel method of in vitro cell surface pH imaging by using a membrane-anchored pH probe, poly(ethylene glycol)-phospholipid conjugated with fluorescein isothiocyanate (FITC-PEG-lipid). When added into the cell culture medium, FITC-PEG-lipid is spontaneously inserted into the plasma membrane via its phospholipid moiety, and retained at the extracellular surface. The ratiometric readout of its fluorescence was unique to the extracellular pH in the range of weakly alkaline and acidic pH. Our study demonstrated that FITC-PEG-lipid is useful as a sensitive and reversible cell-surface-anchored pH probe. The simple cell-surface labeling procedure of FITC-PEG-lipid is advantageous especially when considering its application to high-throughput in vitro assay. Furthermore, PEG-lipid holds a great potential as the membrane anchor of various analytical probes to approach the juxtamembrane environments.

Gastric proton pump (H⁺,K⁺-ATPase) consists of two subunits, a catalytic α-subunit and a glycosylated β-subunit (βHK). So far, properties of the individual carbohydrate residues of βHK have been unclear. Here, we succeeded in visualizing the sialylation and desialylation dynamics of βHK using a fluorescence bioimaging nanoprobe that specifically detects sialic acids. The fluorescence of the probe was observed at the cell surface of H⁺,K⁺-ATPase-expressing living LLC-PK1 cells but not in non-expressing cells. The fluorescence and H⁺,K⁺-ATPase activity in the cells were significantly decreased by sialidase and acidic solution. In gastric mucosa of rats and hogs, the fluorescence of the probe was observed in the samples treated by famotidine, an H₂ blocker, but not by histamine, an acid secretagogue. H⁺,K⁺-ATPase activity in the famotidine-treated samples was significantly higher than the histamine-treated samples. These famotidine-induced effects were weakened by sialidase. Our studies using the nanoprobe uncover a novel negative-feedback mechanism of H⁺,K⁺-ATPase in which sialic acids of βHK positively regulates H⁺,K⁺-ATPase activity, and acidic pH decreases the pump activity by cleaving sialic acids of βHK.

Local metabolite and ionic strength are important factors for maintaining the functions of living cells. We have developed micro-nanoscale electrode and electrochemical sensor based scanning probe microscopy to measure the spatial electrochemical metabolite and ion concentration profile near the sample surface with nanoscale resolution. Scanning electrochemical microscopy (SECM) uses an ultramicroelectrode as a probe for detecting electroactive chemical species (oxygen, ATP, and reactive oxygen species (ROS), and neurotransmitter). SECM has been recognized as an effective tool for investigating micrometer-scale local chemical flux. Miniaturization of the electrode is an important factor for improving SECM resolution. Electrode–sample distance control is also an important factor for measuring fast chemical flux and improving SECM resolution. Distance control by ion current feedback is a promising way for the non-contact investigation of soft materials, SECM–scanning ion-conductance microscopy (SICM) has been used in a hybrid system to improve SECM resolution by controlling the electrode probe and sample distance in solutions without direct contact. SICM is also useful for detecting the ion concentration profile and charge measurement in a solution. We measured the 3D chemical and ion current using SECM–SICM. In this presentation, we report the SICM topography images of gastric surface mucous cell lines (GSM06), which produce periodic acid-schiff and concanavalin A positive glycoproteins. To visualize the damage process of the mucosal layer, we added ethanol to GSM06 cells and imaged the topography change using SICM. We also performed topography and electrochemical simultaneous imaging using SICM-SECM to identify the mucosal layer without labelling.

Continuous and real-time measurement of local concentrations of systemically administered drugs in vivo must be crucial for pharmacological studies. Nevertheless, conventional methods require considerable samples quantity and have poor sampling rates. Additionally, they cannot determine how drug kinetics correlates with target function over time. Here, we describe a system with two different sensors. One is a needle-type microsensor composed of boron-doped diamond with a tip of ∼40 μm in diameter, and the other is a glass microelectrode. We first tested bumetanide. This diuretic can induce deafness. In the guinea-pig cochlea injected intravenously with bumetanide, the changes of the drug concentration and the extracellular potential underlying hearing were simultaneously measured in real time. We further examined an antiepileptic drug lamotrigine in the rat brain, and tracked its kinetics and at the same time the local field potentials representing neuronal activity. The action of the anticancer reagent doxorubicin was also monitored in the cochlea. This microsensing system may be applied to analyze pharmacokinetics and pharmacodynamics of various drugs at local sites in vivo, and contribute to promoting the pharmacological researches.

With our social change toward a 24-hour society, a substantial proportion (ca. 27%) of workers is engaged in shift-work schedules. However, epidemiological studies have indicated that such shift-work schedules increase the risk of obesity, cancers, diabetes, and cardiovascular diseases. Circadian clock-related disorders are now prevalent in our society. In mammals, circadian rhythms in physiological function are regulated by a molecular oscillator consisting of circadian clock genes. Disruption of cellular circadian rhythms is well-recognized to be associated with cancer development and tumorigenesis; however, the underlying mechanism is not fully understood.

　Solid tumors are composed of phenotypically and functionally heterogeneous cells. Among them, highly tumorigenic cancer stem cells (CSCs) generate intermediate progenitors and terminally differentiated cells. Similar to physiological stem cells, CSCs often exhibit resistance to various chemotherapeutic drugs. Recently, we found that oncogenic transformation of circadian clock-defective cells exhibited CSC phenotype cells. Furthermore, oncogenic-transformed circadian clock-defective cells also resisted against the cytotoxicity of chemotherapeutic drugs.

　In this symposium, I would like to summarize our recent findings on the underlying mechanism of development of chemoresistance in circadian clock-defective cells, and also to introduce the chronopharmacological strategy for treatment of malignant tumors.

Vitamin K (VK) is a fat-soluble vitamin involved in the regulation of blood coagulation. Mammals do not synthesize VK and must therefore obtain this vitamin by intestinal absorption. The molecular mechanism(s) of the VK absorption process are not clear. Based on the known role of the NPC1L1 protein in the intestinal absorption of fat-soluble compounds such as cholesterol and vitamin E, the possible uptake of VK via an NPC1L1-mediated pathway was examined. In vitro studies using NPC1L1-overexpressing cells and in vivo studies revealed that intestinal VK absorption is NPC1L1-dependent and inhibited by ezetimibe, an NPC1L1-selective inhibitor. In addition, in vivo pharmacological studies demonstrated that the co-administration of ezetimibe and warfarin, a VK antagonist used as an anticoagulant drug, caused a reduction in hepatic VK level and enhanced the pharmacological effect of warfarin. These adverse events caused by the co-administration were rescued by oral VK supplementation, suggesting that the drug interaction effects observed were the consequence of ezetimibe-mediated VK malabsorption. This non-idiosyncratic drug interaction mechanism was supported by clinical results showing that in the majority of warfarin-treated patients, anticoagulant activity was enhanced by co-treatment with ezetimibe. These findings suggest a novel mechanism of drug-drug interaction mediated by the alteration of the kinetics of essential vitamins. In the presentation, we are going to introduce the physiologically, pharmacologically, and clinically important topic with recent progress.

1) Takada T, et al., Science Translational Medicine. 7:275ra23, 2015.

2) M Ito S, et al., Circulation Journal. accepted.

Research on human genetics of schizophrenia enables to discover effective targets for its diagnosis and medication because of its high heritability. Rare or de novo copy-number variations (CNVs) are likely the most significant contributors to the pathogenesis of schizophrenia. We recently found novel schizophrenia-associated CNVs including ARHGAP10. ARHGAP10 is a member of the Rho GTPase-activating protein (RhoGAP) family that contributes to organizing the actin skeleton, as well as neuronal polarization. We developed a mouse model of schizophrenia patient with both a deletion-type CNV and a single-nucleotide variation (SNV) in the RhoGAP domain of ARHGAP10 gene (ARHGAP10 mutant mice). The mutant mice showed emotional abnormality and potentiation of psychostimulant-induced hyperlocomotion. Furthermore, psychostimulant-treated ARHGAP10 mutant mice showed a marked reduction of percentage of accuracy compared with psychostimulant-treated wild-type mice as well as saline-treated ARHGAP10 mutant mice in a translatable visual discrimination task that reflects cognitive function. These findings suggest that mutations in ARHGAP10 increase the risk of schizophrenia, and Rho signaling pathway may be a potential therapeutic target to develop novel antipsychotics.

With innovative advancements in science & technology, cancer treatment has dramatically improved by discovering molecular targeted agents. However, identifying eligible patients and predicting their therapeutic effects still remain a great challenge. Because genetic and molecular differences of tumors significantly affect therapeutic effects in clinical, establishing individualized medicine based on precise molecular pathogenesis is urgently required.

Cylindromatosis (CYLD) was originally identified as a tumor suppressor because loss of which causes a benign human tumor called cylindromatosis. Increasing clinical evidence reveals that dysfunction of CYLD by loss of its expression is believed to play key roles in diverse pathological processes in various types of tumors. Moreover, our results have shown that loss of CYLD expression not only be involved in tumor malignant transformation, but also serves as a prognostic & predictive biomarker for several tumors.

In this session, we focus on the clinical significance of CYLD and introduce our approaches toward developing a novel molecular targeted therapies. Deeper understanding of more biological feature and clinical significance of CYLD may open up novel strategies for establishing individualized cancer treatment for malignant tumors.

Disease-specific iPS cells have been considered and used as platforms for disease modelinsg and drug discovery for intractable diseases. In the field of cardiovascular medicine, iPS cells have been generated from patients with heart diseases including inherited cardiomyopathy. The disease-specific iPS cells showed reproduce the certain parts of phenotype of the disease on culture dishes in in vitro systems, but the cells do not necessarily recapitulate patients clinical properties, particularly those of physiological-/pathophysiological aspects. The point should be solved to establish disease reliable platforms. The discrepancy may be attributed to the lack of developmental process during culture procedure. To settle the problems, various techniques have been attempted such as culture dishes with specific structures. 

In this symposium, introducing the phenotype of disease specific iPS-cells from patients with cardiomyopathy, we will discuss what need to be done to reproduce "human hearts on culture dishes".

Since human induced pluripotent stem cell derived cardiomyocytes (hiPS-CMs) became available, its usefulness for safety evaluation aiming at improving predictability has been actively examined in many facilities. Among them, the researches focusing on drug induced ventricular arrhythmias using hiPS-CMs have been validated. On the other hand, drug related supraventricular arrhythmias including atrial fibrillation and sinus node dysfunction, which are increasing year by year in clinical, might not be detected by existing methods using hiPS-CMs because the cells being used are mainly constituted of ventricular myocytes. Currently, we are trying to establish the cardiotoxicity system using hiPS cell derived atrial myocytes, and some differences between atrial and ventricular myocytes in drug response are being acquired. In this symposium, we will introduce some evidence and discuss the usefulness of evaluation system using hiPS atrial myocytes as a detection method of supraventricular arrhythmia.

Scattering light has an interesting and important feature. There are various kinds of interaction between light and molecule, and the scattered light includes internal information of the molecule. Raman scattering inheres all the vibration mode of molecular bonds composing a molecule, and second harmonic generation (SHG) light, which is one of second-order non-linear scattering light, is derived from electric polarizations in the molecule, in other words, includes structural information in protein.

While states of cell are usually defined by protein/gene expression patterns, we have proposed applying Raman spectra to a cellular fingerprinting as an alternative for identifying the cell state, and now succeeded in predicting gene-expression of antibiotic bacteria in combination with machine learning technology. Meanwhile, SHG microscopy has been used to visualize fiber structures in living specimens, such as collagen, and microtubules as a label-free modality. By utilizing the feature that SHG senses protein structure change, we developed a new method to measure actomyosin activity in cardiac cells. The most important advantage of use of the scattering light is their non-labeling and non-invasive capability.

Deep learning technology is rapidly advancing, and is now used to solve complex problems. induced pluripotent stem cells (iPSCs) can be used for several purposes such as regenerative medicine, disease modeling study and drug screening. It is inevitable to identify iPSC-derived differentiated cells in microscopy for any use. Here, we used deep learning to establish an automated method to identify endothelial cells derived from iPSCs, without the need for immunostaining or lineage tracing.

Since the identification of endogenous H₂S in the mammalian brain in 1989, studies of this molecule uncovered physiological roles in processes such as neuromodulation, vascular tone regulation, cytoprotection against oxidative stress. We previously demonstrated that H₂S induces Ca²⁺ influx in astrocytes by activating transient receptor potential (TRP) channels. During this study we found that H₂S_n activates TRP ankyrine 1 (TRPA1) channels much more potently than does H₂S and that 3-mercaptopyruvate sulfurtransferase (3MST) produces H₂S₂ and H₂S₃. Recently, we demonstrated that the chemical interaction of H₂S with nitric oxide (NO) generates H₂S₂ and H₂S₃ that may be a mechanism of a synergistic effect between H₂S and NO we previously showed in the regulation of vascular tone. We showed that cysteine persulfide (Cys-SSH) together with its glutathione (GSH) counterpart (GSSH), both of which have been proposed to be involved in redox homeostasis, are also produced by 3MST. We will show our recent observation that sulfite protects neurons from oxidative stress more efficiently than H₂S and H₂S_n with a distinctive mechanism.

For detailed studies of the physiological functions of reactive sulfur species (RSS) such as H₂S and sulfane sulfur, we set out to develop a highly selective and easy-to-use fluorescence probe for H₂S. We designed and synthesized a novel fluorescence probe for H₂S, HSip-1 (Hydrogen Sulfide imaging probe-1), utilizing macroazacyclic complex chemistry with copper ion (II) (J. Am. Chem. Soc. 133, 18003-18005 (2011)). HSip-1 showed the fluorescence increase (by 50 fold) within several seconds upon addition of 10 μM H₂S, whereas almost no fluorescence increment was observed upon addition of 10 mM GSH. HSip-1 also showed high selectivity over other biothiols, ROS, and RNS. We could also visualize H₂S in HeLa cells with HSip-1 DA (a cell-membrane permeable derivative of HSip-1) upon addition of Na₂S. Moreover, we applied HSip-1 to the detection of enzymatic activity of H₂S-producing enzymes in vitro. We successfully monitored the time-dependent H₂S production by 3-mercaptopyruvate sulfurtransferase (3MST) and cystathionine γ-lyase (CSE). We then applied HSip-1 to the inhibitor high-throughput screening (HTS) of a chemical library containing 170,000 compounds from The University of Tokyo, Drug Discovery Initiative, and found selective inhibitors for 3MST and CSE (Sci. Rep. 7:40227 (2017)). We also recently developed a fluorescence probe for sulfane sulfur (Chem. Commun., 53, 1064-1067 (2017)).

Electrophysiological, pharmacological and gene-knockdown studies have shown that hydrogen sulfide (H₂S) promotes pain or itch by enhancing Ca_v3.2 T-type Ca²⁺ channel activity. We thus examined the effect of Ca_v3.2 gene deletion on H₂S-dependent somatic or visceral pain and itch. In wild-type mice, intraplantar and intracolonic administration of Na₂S, an H₂S donor, caused somatic and colonic pain/hypersensitivity, respectively, and intradermal injection of Na₂S in the cheek evoked both pain and itch responses. These responses to Na₂S challenge disappeared in Ca_v3.2-KO mice. Ca_v3.2 deletion did not affect the partial sciatic nerve ligation (PSNL)-induced neuropathic allodynia in mice, but removed the anti-allodynic activity of T-type Ca²⁺ channel blockers. On the other hand, Ca_v3.2 deletion abolished endogenous H₂S-dependent bladder pain in mice with cyclophosphamide-induced cystitis, and the butyrate-induced colonic hypersensitivity in mice, models for bladder pain syndrome (BPS) and irritable bowel syndrome (IBS), respectively. Our data thus suggest that Ca_v3.2 plays a key role in exogenous H₂S-induced pain and itch, and in visceral pain signaling in BPS and IBS models, although unknown neuronal systems might compensate Ca_v3.2 deficiency in PSNL-induced neuropathy.

Hydrogen sulfide (H₂S) is known as a toxic gas, but has been focused as a biological mediator, which modulates signal transduction and protects cells and tissues from oxidative stress. Endogenous H₂S is mainly generated from L-cysteine, while a novel biogenesis pathway of H₂S from D-cysteine has been recently identified. In this pathway, D-amino acid oxidase (DAO) converts D-cysteine to 3-mercaptopyruvate (3MP), followed by the generation of H₂S from 3MP by 3-mercaptopyrvate sulfurtransferase. DAO is especially abundant in cerebellum among various brain regions and mediates efficient generation of H₂S from D-cysteine in the cerebellar tissues. Cerebella Purkinje cells (PCs) are characterized by the highly-branched dendrites and are important for cerebellar functions. The dendritic shrinkage and degeneration of PCs are frequently observed in patients and model mice of cerebellar ataxias. We revealed that D-cysteine enhances dendritic development of primary cultured PCs, but L-cysteine does not. This effect was inhibited by DAO inhibitors and reproduced by 3MP and a H₂S donor, suggesting that this enhancement by D-cysteine is caused by the production of H₂S. Taken together, D-cysteine would be available as a neuroprotectant against cerebellar ataxias.

Brain ischemic tolerance is an endogenous neuroprotective mechanism, whereby an experience of non-lethal ischemic episode (preconditioning; PC) produces resilience to subsequent lethal ischemia. We previously showed that PC caused activation of astrocytes and a subsequent upregulation of P2X7 receptors, activation of which induced ischemic tolerance via upregulation of HIF-1α in astrocytes. P2X7 receptor requires relatively higher extracellular ATP concentrations (ATPo) for its activation. However, the PC-evoked increase in ATPo was not enough to activate P2X7 receptor. Here, we show that astrocytes have a unique mechanism of P2X7 receptor activation with lower ATPo, thereby leading to ischemic tolerance. It has been reported that NAD at lower ATPo could induce prolonged activation of P2X7 receptor via ART2-catalyzed ADP-ribosylation. Thus, we tested effect of NAD on astrocytic P2X7 receptors, and found that NAD increased HIF-1α in WT astrocytes but not in P2X7 receptor-deficient astrocytes in vitro. We also found that ART2 is selectively expressed and upregulated by PC in astrocytes. Taken together, our findings suggest that astrocytes could activate P2X7 receptors by their unique mechanisms, i.e., NAD/ART2/P2X7 signal pathways and induce ischemic tolerance.

During the purinergic chemical transmission, neurons, neuroendocrine cells, glial cells, immune cells and other types of cells secrete ATP, and communicate with each other through purinoceptors on the plasma membrane. In spite of well-understood features on the signaling cascade after stimulation of the purinoreceptors, the mechanism of how ATP is stored and released from the purinergic cells is far less characterized. In this study, we focus on the mechanism on vesicular secretion through vesicular nucleotide transporter (VNUT). VNUT transports nucleotides such as ATP and ADP and plays an essential role in the vesicular storage and secretion of ATP. Recently, we identified the clinically available VNUT inhibitor clodronate, which impaired vesicular ATP release from neurons, microglia, and neutrophils. Clodronate also impaired neutrophil migration and attenuated neuropathic and inflammatory pain in vivo. Although more extensive works will be necessary, these studies show that VNUT-specific inhibitor is able to control vesicular ATP release in vivo and that VNUT regulates purinergic chemical transmission.

Extracellular nucleotides can control proliferation of neural stem cells (NSCs). However, their effects on selection of neuronal subtypes have not been elucidated. Glutamatergic neurons are the most abundant subtype in the mammalian brain. Production of this neuronal subtype can be observed not only in the development of the forebrain, but also through life in the hippocampus. In the development, glutamatergic neurons are produced from neuroepithelial cells at the dorsal side of the anterior neural tube. In the adult hippocampus, stem cells located at the sub-granular zone can produce glutamatergic granule neurons through life. We found that the expression of the nucleotide receptor P2Y4 was transiently augmented in the course of neuronal differentiation of mouse ES cells. Interestingly, a subpopulation of type 2 NSCs of the adult mouse hippocampus also expressed P2Y4. The activation of P2Y4 in those cells increased proportion of glutamatergic subtype in their descendant neurons. Our results provide evidence that differentiating NSCs pass through a stage in which nucleotides can affect subtype marker expression of their descendant neurons.

Although psychotropic drugs act on neurons and glial cells, how glia respond, and whether glial responses are involved in therapeutic effects are poorly understood. Here, we show that fluoxetine (FLX), an anti-depressant, mediates its anti-depressive effect by increasing the gliotransmission of ATP. FLX increased ATP exocytosis via vesicular nucleotide transporter (VNUT). FLX-induced anti-depressive behavior was decreased in astrocyte-selective VNUT-knockout mice or when VNUT was deleted in mice, but it was increased when astrocyte-selective VNUT was overexpressed in mice. This suggests that VNUT-dependent astrocytic ATP exocytosis has a critical role in the therapeutic effect of FLX. Released ATP and its metabolite adenosine act on P2Y₁₁and adenosine A2b receptors expressed by astrocytes, causing an increase in brain-derived neurotrophic factor in astrocytes. These findings suggest that in addition to neurons, FLX acts on astrocytes and mediates its therapeutic effects by increasing ATP gliotransmission.

Many strategies have been tried to produce monoclonal antibodies (mAbs); however, there have been several problems about focusing on molecular targets and screening methods. For instance, the high tumor/normal ratio of antigen expression using DNA microarray has been thought to be important when we determine the molecular targets for antibody-drug. Although many antigens are expressed highly in tumors, those antigens have been removed from the candidates of antibody-drug targets because they were also expressed in normal tissues. We recently established a novel technology to produce a cancer-specific monoclonal antibody (CasMab). The post-translational difference such as glycans can be utilized to produce the CasMab, although the protein possesses the same amino acid sequence in both cancer and normal cells. We have already produced CasMabs against several glycoproteins such as podoplanin, which is expressed in both cancer and normal cells. Those CasMabs possess antibody-dependent cellular cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC) in vitro and anti-tumor effect in xenograft models in vivo. In conclusion, the CasMab technology is the platform to develop cancer-specific mAbs, which could attack only cancer cells without side effects.

Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response to infection. Neutrophil-associated inflammation or microthrombus formation (Immunothrombosis) in the lung resulted in acute respiratory distress syndrome, the most important cause of death in septic multiple organ failure. Histidine-rich glycoprotein (HRG) is a 75 kDa glycoprotein mainly produced by liver. HRG is known as the plasma factor to regulate coagulation/fibrinolysis, immune response and angiogenesis. Our recent studies revealed that plasma HRG levels significantly decreased in cecal ligation puncture (CLP) septic mice model and administration of HRG dramatically improved the survival rate of CLP mice associated with the inhibition of immunothrombosis and neutrophil extracellular trap formation in pulmonary vasculatures by keeping neutrophils quiescent morphologically and functionally, and the protection of vascular endothelial cells and inhibition of erythrocyte aggregation. Thus, HRG-supplementary therapy may provide a novel strategy for the treatment of septic patients.

Positron emission tomography (PET) is a powerful tool for drug discovery because it would enable the evaluation of target engagement (proof-of-concept), pharmacokinetics, and therapeutic efficacy of drug candidates in humans using PET radiopharmaceuticals. Recently, biopharmaceuticals are attracting increasing interest because they possess not only high binding affinity and specificity but also creates unique actions to attack pathologic lesions, requiring the radiolabeling methods of proteins to evaluate their pharmacokinetics and efficacy. In this presentation, we review the radiolabeling methods for the biopharmaceutical itself using long-half lived PET radionuclides such as Cu-64 and Zr-89, which are suitable for the evaluation of their pharmacokinetics. Since Cu-64 and Zr-89 showed relatively high radiation, shorter half-lived radionuclides such as F-18 is better from the point view of radiation. Here, we show our recent developed approach for the production of F-18 labeled proteins, which are suitable for the evaluation of their therapeutic efficacy and patient selection with minimum radiation.

Previously, we showed that spinal pituitary adenylate cyclase-activating polypeptide (PACAP)/PAC1 receptor signaling triggers long-lasting pain-like behaviors through astroglial activation. Since astrocyte-neuron lactate shuttle (ANLS) could be essential for long-term synaptic plasticity, we aimed to elucidate a possible involvement of spinal ANLS in the development of the PACAP-evoked pain-like behaviors. A single intrathecal administration of PACAP induced short-term spontaneous aversive behaviors, followed by long-lasting mechanical allodynia in mice. These behaviors were inhibited by DAB, an inhibitor of glycogenolysis, and this inhibition was reversed by simultaneous L-lactate application. In the cultured spinal astrocytes, the PACAP-evoked glycogenolysis and lactate secretion were inhibited by a protein kinase C (PKC) inhibitor, and the PKC inhibitor attenuated the PACAP-induced pain-like behaviors. Moreover, an inhibitor for the monocarboxylate transporters blocked the lactate secretion from the spinal astrocytes and inhibited the PACAP-evoked pain-like behaviors. In this symposium, we will further discuss possible involvement of the spinal PACAP-ANLS signaling in an experimental model of neuropathic pain.

Astrocytes play a key role in the maintenance of synaptic transmission by producing L-lactate via the astrocyte-neuron lactate shuttle (ANLS). Astrocyte activation in the spinal cord is involved in the expression of neuropathic pain. These reactive astrocytes are suggested to play an important role in the maintenance of neuropathic pain. We investigated the role of the ANLS in the spinal cord on hyperalgesia in neuropathic pain in mice. We also investigated the cellular mechanisms of spinal L-lactate-induced mechanical hyperalgesia. We revealed that the selective activation of spinal dorsal horn astrocytes causes mechanical hyperalgesia through the excessive L-lactate supply to neurons via monocarboxylate transporters (MCTs). We also found that L-lactate transported into neurons may produce mechanical hyperalgesia. In addition, application of L-lactate enhanced the excitatory neurotransmission evoked by mechanical stimulation in the dorsal horn of spinal cord. Moreover, intrathecal treatment with L-lactate also activates protein kinase A (PKA) signaling. These results suggest that the enhanced ANLS sensitizes the nociceptive transmission in the spinal cord through the activation of PKA signaling.

The excitatory transmission from the solitary tract (TS) primary afferents to the second-order neurons in the nucleus of the solitary tract (NTS) depends largely on the energetic supply through monocarboxylate transporters (MCTs) (Nagase et al., 2014). We examined whether this large dependency of the excitatory transmission on lactate supply observed in the TS-NTS synapse is commonly shared by brain circuits underlying pain as "sensory and emotional experience". The lateral parabrachial nucleus (LPB) is the site where the nociceptive information arising from the spinal dorsal horn and trigeminal nerve converges and functions as a relay of these signals to the pain-associated networks. The central amygdala (CeA) is the most important target of the ascending LPB projections, which undergoes robust synaptic potentiation in various pain models (Kato et al., 2018). In brain slices prepared from rats and mice, the excitatory synaptic transmission from the LPB to the CeA was significantly attenuated by inhibition of MCTs with 4-hydroxycinnamic acid (4-CIN) without significant changes in paired-pulse ratio. This effect was similarly observed in setups where LPB fibers were stimulated electrically and optogenetically. Interestingly, in the current clamp recordings, blockade of MCTs resulted in postsynaptic depolarization in the CeA neurons, unlike in the pyramidal neurons in the lateral amygdala, hippocampal CA1 and Purkinje neurons in the cerebellum, which were hyperpolarized by activation of K_ATP channels. As the excitability of CeA neurons is a crucial determinant of the emotional/aversive aspects of pain, the pharmacological regulation of the lactate transport in the CeA would be a candidate as a target for resetting aberrantly augmented nociception-emotion link and malfunctioning descending regulation.