Studying drug actions often takes you to new, completely unexpected, research avenues. This is joy of pharmacology. I first became aware of pharmacology as such science when I worked as a postdoctoral fellow in John Vane's laboratory at Wellcome Research Laboratories in England, where people used bioassay, blocked the actions of known substances with respective antagonists and searched for new bioactive substances. I also experienced drug development process there, because Wellcome Research Laboratories were the research institute of the Burroughs-Wellcome Company. I realized that the whole process of drug development from basic research to clinical trial is an enormously big scale of experiment. My research thereafter on the prostanoid receptors and Rho GTPases both originated from the analysis of drugs (compounds and toxin, respectively), and took me to enjoy exploration of almost all body functions and in the world of cell biology. Quite recently, I have been associated with research collaboration with Astellas Pharma and have been using drug candidates and enjoying research avenues new to me and new to all. Joy of pharmacology never ceases.

Immune responses are accompanied by dynamic changes in gene expression. Many transcription factors including NF-κB and AP-1 are involved in induction of genes involved in inflammatory and immune responses. However, recent studies have revealed that control of gene expression at the mRNA level is as important as transcriptional control in the immune response. We have shown that Regnase-1 encoded by the Zc3h12a gene is an endoribonuclease involved in destabilization of a variety of mRNAs including IL-6,IL-12, and Regnase-1 itself mRNAs via the stem loop structure present in the 3'UTR of these genes

Although originally identified as LPS-inducible gene, Regnase-1 protein is present in unstimulated cells, and disappears in response to Toll-like receptor ligands via an IKK-dependent proteasome degradation pathway or in response to T cell receptor stimulation through the cleavage by Malt-1. Thus, Regnase-1 acts as a brake in unstimulated cells as well as a negative feedback regulator after cellular activation. Recently we found that IL-17 signal also inhibits the function of Regnase-1. I would like to discuss the role of Regnase-1 in the immune response.

Whole-brain imaging and systems analyses of entire brains at subcellular resolution and subsequent processing of the resulting images are prerequisites to investigate anatomical and functional brain networks to understand their function and dysfunction. However, it is still a challenge due to the trade-offs between imaging speed and spatial resolution irrespective of if tissue clearing methods are used. To overcome the issue, we have been attempting to increase the imaging throughput and relieve bottlenecks in the procedure, and have recently developed an automated high-speed imaging system for block-FAce Serial microscopy Tomography (termed FAST). By using this system, it became possible to perform quantitative comparisons of whole-brain structures and neuronal activities at the cellular level using the spatial coordinated alphanumeric data of brain cells and pattern recognition methods. The FAST system thus paves the way for imaging analyses of the brain and provides new opportunities for unbiased and hypothesis-free approaches that contributes to investigate molecular mechanisms and therapeutic drug targets for brain disorders.

Protein toxins from venomous organisms have been valuable tools for investigating the structure and gating mechanisms of voltage-activated ion channels. Transient Receptor Potential (TRP) channels are a large family of ion channels that are activated by diverse stimuli and ligands, including second messengers, temperature, voltage and natural products such as capsaicin, menthol and wasabi. We have begun to investigate the structure and gating mechanisms of the heat-activated TRPV1 channel using the double-knot toxin (DkTx) from tarantula venom. I will present the structure of DkTx that we solved using NMR, and show how we have docked DkTx into the electron density maps from the recent single particle EM structure of the toxin bound TRPV1 channel to reveal a range of interesting features of the toxin-channel interaction. In particular, our results reveal that DkTx binds to the perimeter of the external pore of TRPV1 at the interface of the channel with the surrounding lipid membrane. I will also talk about functional experiments suggesting that DkTx and extracellular ions profoundly alter activation of TRPV1 by heat, implicating the external pore in the mechanism of temperature-sensing.

We have developed a next-generation proteomics approach—in vitro proteome–assisted multiple reaction monitoring for protein absolute quantification (iMPAQT)—that allows genome-wide absolute quantification of the human proteome and is reliant on the production of ～18,000 recombinant proteins. We applied iMPAQT to delineate the metabolic landscape of human diploid fibroblasts. Oncogenic transformation of these cells gave rise to relatively small but global changes in metabolic pathways that account for aerobic glycolysis (Warburg effect) and increased rates of macromolecule synthesis. Modulation of metabolic enzyme expression revealed an unexpected functional interaction between glycolysis and the pentose phosphate pathway that facilitates nucleic acid synthesis. Furthermore, integration of proteomic and metabolomic data allowed construction of a mathematical model for identification of key enzymes responsible for the metabolic shift in cancer. We found that substantial remodeling in glutamine metabolism, which we call the "second" Warburg effect, is essential for malignant phenotypes of cancer cells. Our results thus provide a global view of metabolic restructuring in cancer that underlies adaptation to a rapid growth state.

Plant cells have specific organelle distinct from moving animals, vacuoles, which store toxic chemicals such as ferric ions. Vacuolar iron transporter 1 (VIT1) transports cytoplasmic ferric ions into vacuoles. We solved crystal structure of VIT1, which acts as an H⁺-dependent antiporter for Fe²⁺ and other transition metal ions. VIT1 adopts a novel protein fold forming a dimer of five-membrane-spanning domains, forming an ion-translocating pathway constituted by the conserved methionine and carboxylate residues at the dimer interface. The second transmembrane helix protrudes from the lipid membrane by about 40 Å and forms a three-helical-bundle triangular cytoplasmic domain, which binds to the substrate metal ions and stabilizes their soluble form, thus playing an essential role in the transport.

Recent progress in cryo-EM single particle analysis enables us to solve large membrane protein structure rapidly. I would like to present here new structures of heterodimeric amino-acid transporter and two physical stimuli-sensing channels from human to uncover their molecular mechanisms.

By combining springs with motors and chemical switches biological systems can achieve great scales of mechanical power amplification, for example when jumping or launching a projectile. However, the molecular mechanisms of how this is achieved remain unknown. The giant muscle protein titin, composed of hundreds of tandem Ig domains, is a complex elastic protein whose role in muscle function is still poorly understood. A crucial recent discovery that we have made is that titin domains do a surprisingly large amount of mechanical work when they fold against an opposing mechanical force. We have shown that the amount of mechanical work done by a folding titin Ig domain can be 2-3 times larger (～120 zJ) than that of the chemically powered motor myosin II; ～38 zJ. Titin molecules store mechanical energy by unfolding and extending under force. Elastic energy is stored this way by stretching caused by gravitational pulling during locomotion, inertia, chemical modifications, and ATP powered sources to name a few. Titin unfolding occurs at varying rates over a very wide range of forces above >8 pN. By contrast, most of the stored mechanical energy is delivered back only over a small range of forces where the folding probability increases from 0 to 1 (< 6 pN) and the folding protein does large amounts of mechanical work. Thus, protein folding/unfolding is likely to operate as a mechanical battery where different types of energy sources are stored, and then converted back into contractile power.

Given that titin is now known to be the third filament of muscle, determining if protein folding can deliver work quickly enough to match the power output of the myosin motors, is a central question to be answered. The mechanical power output of protein folding is a novel concept and thus has never been studied before.

Cryptic cysteine residues are common in the elastic I band region of titin where they can oxidize to form intra-domain disulfide bonds, limiting the extensibility of an unfolding Ig domain.

Here we use magnetic tweezers force spectroscopy to study the folding dynamics of a disulfide bonded modular titin protein operating in the physiological range, with the ability to control the oxidation state of the protein in real time. We show that the midpoint folding probability of the parent Ig domain reversibly shifts up from 4.0 pN to 12.8 pN upon oxidation. In this force range, the folding contraction dominates the elastic recoil of the protein, delivering stepwise mechanical work which depends on the oxidation state in an all-or-none manner. For example, the output power of a folding contraction at 6 pN goes from 0 zW to 6,000 zW upon introduction of the disulfide bond. This large amount of power is delivered by folding at forces where single molecular motors are typically stalled. From our observations we predict that during muscle contraction, activation of myosin II motors by Ca⁺⁺ leads to a drop in the force experienced by titin, triggering delivery of mechanical power by titin folding. Thus, it seems inevitable now that the three filaments of the muscle sarcomere act in concert to both store and deliver mechanical power, revolutionizing our understanding of the molecular mechanisms of muscle contraction.

Neurotransmitter transporters present at the cell surface are well-established asthe primary targets for psychostimulant drugs of abuse and for drugs such asmethylphenidate and amphetamines, which are used to treat attention deficitdisorders. In recent studies, we have observed that once amphetamines enterneurons they can activate multiple intracellular signaling pathways. Within thecell, amphetamines activate the small GTPases, Rho and Rac1, and triggerendocytosis of the dopamine transporter (DAT) and a neuronal glutamatetransporter (EAAT3) by a RhoA-dependent internalization pathway. These eventsdepend upon the expression of an intracellular G-protein coupled trace aminereceptor (TAAR1) that signals through at least two types of G-proteinalpha-subunit within the cell. Using a series of subcellularly-targeted geneticfluorescence resonance energy transfer (FRET) sensors to detect RhoA or PKAactivation, we have been able to characterize the subcellular membranecompartments where TAAR1 signaling events initiate. These results imply thatamphetamine-like drugs not only inhibit monoamine transport and potentiateneurotransmitter action, but they also activate signaling pathways through theirdirect action on an intracellular GPCR target. This lecture will highlight therole of TAAR1- and other GPCR-mediated signaling events in amphetamine actionand will consider how they are linked to the action of a variety of drugs thatmodulate monoamine signaling.

Focusing on melatonin secreted from the pineal body of the brain, melatonin receptor agonist Ramelteon (Product name: Rozerem) which was totally different from conventional hypnotics was discovered in Japan. Drug discovery does not come true overnight. Starting with disease and target selection, construction of screening system, drug design, optimization study, toxicity testing, clinical trials and many years of trial and error are repeated tremendously. Only one compound that has been thought out and carefully nurtured by the single-minded many researchers finally reach the goal (product). The burden of the pharmaceutical company is heavy under the limitations of low molecule drug discovery and the exhaustion of drug discovery targets, and unless new strategies to enhance the success rate are taken, the discovery of new medicine can't be hoped at all. In order to continue to maintain world-class drug discovery capabilities and to make drug discovery a reality, aggressive cooperation of industry, government and academia will create highly original results in Japan. I would like to mention how to accomplish the drug discovery through my thorns path leading to drug discovery. I really hope that one grain of drug created by many hard work researchers will present a smile to many patients.

Animals survive by detecting and adapting to the ambient temperatures. One of the important molecules involved in the temperature detection is TRP channel which consists of 28 members in 6 subfamilies in mammals. Eleven among the 28 TRP channels are activated by temperature changes, and thus called ‘thermosensitive TRP channels' Cation influx through temperature-sensitive channels like TRP channels causes some depolarization leads to activation of voltage-gated Na⁺ channels, followed by action potential generation. Because TRP channels have relatively high Ca²⁺ permeability, Ca²⁺ entering the cells activates Ca²⁺-activated Cl^- channels, causing further depolarization. Thermosensitive TRP channels are activated in the different temperature ranges and involved in various physiological phenomena in many species. I will introduce structures and functions of thermosensitive TRP channels together with significance of the complex of TRP channels and Ca²⁺-activated Cl^- channels, anoctamin 1. I will also discuss the TRP channels as potential drug targets. Furthermore, the evolutional aspects of thermosensitive TRP channels will be mentioned since animals are thought to have evolved by changing the expression and function of thermosensitive TRP channels through the changes in ambient temperatures.

Intracellular Ca²⁺ signaling plays obligatory roles in the regulation of cellular physiological functions, and also the proliferation and/or death of cells. Hyper-activation of Ca²⁺ signaling often initiates and/or facilitates the pathological conditions in many kinds of diseases and has been studied as the potential target for new therapeutic approaches and drug discovery. The generation of Ca²⁺ signaling has two major pathways; the Ca²⁺ inflow and the Ca²⁺ release from intracellular stores, which are controlled in extensively different manners depending on cellular excitability. In excitable cells, membrane potential changes due to ion channel activities and Ca²⁺ signaling are bidirectionally regulated to maintain the homeostasis of physiological functions efficiently by Ca²⁺ microdomain formation, which has been revealed by molecular imaging. Even in non-excitable cells, the resting membrane potential substantially modulates Ca²⁺ signaling via the inflow through non-voltage-dependent Ca²⁺ channels, such as store-operated Ca²⁺ entry. The crucial significance of K⁺ channel in Ca²⁺ signaling is attributable to its inverse functions in excitable and non-excitable cells. Based on the large molecular diversity, the heterologous multimer-formation and the tissue-specific expression pattern, K⁺ channels particularly in non-excitable cells are now recognized as a hot target of drug discovery research and development.

Primary cilia are solitary, generally non-motile, hair-like protrusions that extend from the surface of cells between cell divisions. Defects in primary cilia formation and function result in many developmental diseases and some adult ciliopathies. In this lecture, I will discuss the function of primary cilia in neurons, glia, kidney, and cell lines. The focus will be on measurements of ion currents in primary cilia, their underlying physiology, and the structures of these channels.

Members of the nuclear receptor family, the retinoic acid receptors (RARs) alpha, beta, and gamma, regulate cell differentiation and epigenetic states of stem and differentiated cells. We showed that these RARs also play key roles in cancer prevention and in the inhibition of diabetes and hepatic steatosis, but the complexity of signaling by multiple receptors and the regulated production of active ligands make it challenging to establish the molecular mechanisms for these effects. To develop new pharmacological treatments, we generated unique murine models of early stage clear cell renal cell carcinoma (ccRCC) and head and neck cancer (HNSCC). These models reflect molecular and histological changes that occur in human carcinogenesis, providing us with powerful systems in which to test new drugs and increase our understanding of the early stages of carcinogenesis. In our HNSCC model, we are analyzing retinoic acid receptor (RAR) gamma as a tumor suppressor, and we've shown that a RAR gamma selective agonist inhibits the development of oral squamous cell carcinoma. In our ccRCC model we've found novel therapeutic targets. Using both dietary (i.e., high fat diet) and genetic (i.e., db/db mice) models of diabetes and/or hepatic steatosis, we showed that a RAR beta selective retinoid can reduce the pathological features of diabetes and hepatic steatosis. Furthermore, we reported that this RAR beta selective retinoid is useful in treatment of diabetic nephropathy and is cardioprotective. Thus, combining pharmacological studies with representative disease models provides important insights into many common human diseases (metabolic syndrome, cancer, kidney disease, and heart disease), and can lead to new therapeutics to improve human health.

It is my greatest honor to receive the 12^th Setsuro Ebashi Award of the Japanese Pharmacological Society. By establishing an original intravital multiphoton microscopy, I have been studying on ‘real' cellular dynamics with immune and inflammatory systems in intact tissues and organs. Especially I have first succeeded in visualizing in vivo behaviors of different cell types in bones, such as osteoclasts, which led to significant conceptual advances on dynamic bone/immune systems. This research activity with dynamic imaging technology is not only contributing to development of basic biological science in general, but also serving as a novel way for analyzing in vivo pharmacological actions of different emerging drugs. In this lecture I will outline my research so far as well as present my current trial for launching ‘immunopharmacology in Japan to the world', based on our original research trend.

Injury and inflammation causes severe neurological dysfunction that can be partially reversed by spontaneous regeneration of neuronal network in the central nervous system (CNS). Although CNS environment contains the molecules that inhibit axon regeneration, recent researches pointed out that CNS neuronal network can regenerate spontaneously in the animals of some disease models. Because the CNS environment is separated by the peripheral milieu in the presence of the blood-brain barrier, CNS regeneration is thought to be controlled by the CNS microenvironment. However, we found that factors derived from peripheral tissue leak into the CNS after injury and promote remyelination in a murine model of toxin-induced demyelination. We identified that the remyelination is stimulated by the molecules which are expressed by the peripheral tissues, such as pancreas, adipose disuse, skeletal muscle, and heart. Especially, pancreas-derived FGF21 derived proliferation of oligodendrocyte precursor cells (OPCs) through interactions with beta-klotho, an essential coreceptor of FGF21, and promoted remyelination. In this presentation, I would like to show our recent reports that indicate a potentially important role for the peripheral milieu in promoting CNS regeneration.

Long-distance axonal transport of ligand-receptor complexes from the site of endocytosis in the axon to somatodendrite acts as a signaling platform in neurons. However, the endosome-based transportations called "signaling endosomes" and its physiological significance remain to be elucidated. I discovered that retrograde axonal transport of semaphorin3A and its receptors by signaling endosomes regulates dendritic development, specifically enhances AMPA receptor GluA2 localization in dendrites. The retrograde signaling endosomes also induce anterograde delivery of nascent receptors. The axon-derived neurotrophin signaling endosomes are exocytosed to soma surface membrane where they promote anterograde transport of resident naïve receptors. As a positive feedback mechanism, this enhances the neuronal sensitivity to ligand in neurotrophin signaling. Taken together, these data suggest the antero- and retrograde communication via signaling endosomes plays critical roles in neuronal development. Since many neurodegenerative disorders are featured with impaired axonal transport, I start a project that investigates the role of signaling endosomes during the onset of these neurodegenerative disorders.

MID-NET^® is a medical information database that had been established under the governmental project of the Ministry of Health, Labour and Welfare since FY 2011, for a prompt and proper implementation of drug safety measures. PMDA has worked for developing database system, operating data quality management and establishing utilization rules. As a result, MID-NET^® was officially launched in April 2018 and was opened for utilization by the pharmaceutical industry and academia.

MID-NET^® is expected to become a major data source for more scientific and efficient post-marketing studies on drug safety assessment. We hope that MID-NET^® plays an important role for promoting real world data utilization.

At this symposium, I will introduce the outline of MID-NET^® including date quality management, utilization rules and future outlook of MID-NET^®.

FAERS is a public database that accumulates more than 9 million self-reports of adverse events. In nearly half of the cases, multiple drugs are prescribed, so that potential drug-drug interactions are to be analyzed. Focusing on adverse reactions relating to diabetes mellitus (DM) caused by an anti-schizophrenic quetiapine, we found that concomitant use of vitamin D analogs significantly suppresses the occurrence of quetiapine-induced DM in FAERS. Experimental validation revealed that quetiapine acutely caused insulin resistance, which was mitigated by dietary supplementation with cholecalciferol. In an expression database, several genes downstream of insulin receptor were downregulated by quetiapine. Further experiments clarified that a PI3K regulatory protein gene, pik3r1, was downregulated by quetiapine, which was reversed by cholecalciferol in mouse skeletal muscle. In addition, insulin-stimulated glucose uptake into cultured myotubes was inhibited by quetiapine, which was reversed by pretreatment with calcitriol. These results suggest that vitamin D prevents the atypical antipsychotic-induced hyperglycemia and insulin resistance by upregulation of PI3KR1. Until now, we have obtained several combinations of concomitant medications to reduce specific adverse events by FAERS analysis. This new strategy will pave the way for drug repositioning and clarifying unknown disease mechanisms.

Treatment outcomes of cancer patients have improved with progress in oncology medication therapy, but side effects caused by anticancer agents are becoming widespread. Side effects caused by anticancer drugs not only significantly lower the patient's QOL but also often lead to dose reduction or discontinuation of the anticancer drugs. Addressing these side effects is important for improving patient prognosis. Therefore, improvement of the quality of cancer therapy through the development of preventive drugs against anticancer drug-induced side effects is an urgent goal. In recent years, clinical research has been carried out in Japan using large-scale medical information sources such as disease/side effect databases, in order to accurately evaluate the effects of drug used in clinical practice. Research utilizing such a large-scale medical information database can cover various patient parameters and a wide range of observation areas. Therefore, this approach is suitable for conducting clinical research on rare diseases and low-frequency side effects. In this symposium, we will introduce research conducted using drug discovery tools and cell/animal experiments based on a large-scale medical information database to search for preventive agents against anticancer drug-induced side effects, as well as consider future prospects for this approach.

The Health, Care and Educational Information Evaluation Promotion Organization (HCEI) has established a database centered on electronic medical records in collaboration with 160 medical institutions since 2015. As a characteristic of this database, it contains not only data of DPC and receipt already, but also test results and death data. As a result, we can conduct outcome research to measure the effects of drugs and treatments.

Our database is not dependent on vendors and register in the database about 19 million patients. Based on JLAC10, it has standardized 1000 kinds of inspections (with unit / sample classification), and it becomes a useful database for post marketing surveillance etc.

In addition, we have also started a project to collect data in the registry and the data extraction in the randomized controlled trial conducted by the academic society, and we are promoting efforts to eliminate the load of data extraction in the hospitals. Since effective utilization of medical data is a major cornerstone for drug discovery or quality improvement of medical treatment, we would like to continue to develop the database for the promotion of primary and secondary use of medical information.

The rapid advances in high-throughput technologies have facilitated the collection of multilevel omics data. The increasing volume of multilevel omics data continues to create larger and more complex datasets which are publicly available and can be used to generate disease-associated biological networks and to identify potential therapeutic targets within the networks. Further progress in computational methodology combined with improved disease models will facilitate the prioritization of therapeutic targets in the networks. The integration of public omics database, bioinformatics tools, and disease models can provide a strong foundation for deciphering the complex mechanism of various diseases and for data-driven drug discovery. In this symposium, I would like to demonstrate some examples of how the integrative approaches using public omics databases and animal disease models can be exploited to identify potential therapeutic targets for various disorders.

Drug repositioning, or the identification of new indications of drugs (i.e., new applicable diseases), is an efficient strategy for drug development, and it has received remarkable attention in pharmaceutical science. The drug repositioning approach can increase the success rate of drug development and to reduce the cost in terms of time, risk, and expenditure. In this study, we developed novel machine learning methods for automatic drug repositioning in order to predict unknown therapeutic indications of known drugs or drug candidate compounds. We also proposed to use molecular pathways as the therapeutic targets and develop novel computational approach for screening drug candidate compounds. The prediction is performed based on the analysis of various large-scale omics data and molecular interaction networks of drugs, compounds, genes, proteins, and diseases in a framework of supervised network inference. Our results show that the proposed method outperforms previous methods in terms of accuracy and applicability. We performed a comprehensive prediction of new indications of all approved drugs and bioactive compounds for a wide range of diseases defined in the International Classification of Diseases. We show several biologically meaningful examples of newly predicted drug indications for cancers and neurodegenerative diseases. The proposed methods are expected to be useful for various pharmaceutical applications in drug discovery.

We developed a platform for drug repositioning/rescue by using omics data that express the cell-state changes by drug administration, based on the concept of well-known "connectivity map", which the transcriptome data by drug administration is connected to drug efficacy. In addition to connectivity-map approach, the platform is equipped with an original method to estimate the drug efficacy based on the network analyses. Furthermore, the platform is also equipped with a workflow to estimate new stratification markers of the indications newly found by drug repositioning. Note that in principle, the platform type of drug repositioning/rescue can provide solutions of commercialized drugs against any kind of target diseases. We will illustrate our platform performance with some practical applications, especially in terms of the increase of certainty of new findings, at the spot.

The functions of the lower urinary tract (LUT), to store and periodically releaseurine, are dependent on the activity of smooth and striated muscles in theurinary bladder, urethra, and external urethral sphincter. This activity is inturn coordinated by neural circuits in the central nervous system (CNS). Variousneurotransmitters, including acetylcholine, norepinephrine, dopamine, serotonin,excitatory and inhibitory amino acids have been implicated in the CNS regulationof the LUT. Injuries or diseases of the CNS such as cerebral infarction,Parkinson disease, multiple sclerosis and spinal cord injury, as well aspsychological stress or depression, can produce LUT dysfunctions leading tostorage and voiding LUT symptoms such as urinary frequency, urgency, pain andincontinence or inefficient voiding and urinary retention. The recent researchadvancement in the field of CNS neuro-urology has led to the emergence of newconcepts regarding neural control of the LUT and the etiology of LUTdysfunction. In this symposium, I will overview the recent advancement towardsthe identification of disease-related changes in receptor function and newdelivery systems such as gene therapy techniques, which could lead to the futuretreatment of LUT dysfunction. 

Spinal cord injury (SCI) can lead to detrusor overactivity and detrusor-sphincter dyssynergia, which result in inefficient voiding and bladder wall tissue remodeling such as hypertrophy and fibrosis. However, no effective modality for controlling the bladder remodeling is available. Phosphodiesterase type 5 (PDE5) inhibitors and alpha1-adrenoceptor (α₁-AR) antagonists are used for the treatment of male lower urinary tract symptoms with benign prostatic hyperplasia.

In animal study, PDE5 inhibitor or α_1A/D-AR antagonist treatment suppressed the bladder fibrosis after SCI. PDE5 inhibitor might increases the blood flow and prevents bladder ischemia, resulting in the reducing the load in storage state and the suppressing of bladder fibrosis after SCI. Relaxing the bladder neck and proximal urethra after α₁-AR antagonist treatment might decrease the resistance to urine flow from detrusor–sphincter dyssynergia and residual urine volume reduce the load of the bladder wall during both of voiding and storage states. Therefore, treatment with PDE5 inhibitors and α_1A/D-AR antagonists could be effective for neurogenic lower urinary tract dysfunction including bladder remodeling after SCI.

Stress urinary incontinence (SUI) is a common and bothersome problem among middle-aged women. Various animal models of SUI have been developed to study the pathophysiological process involved in SUI, such as vaginal distention, pudendal nerve injury, or ovariectomy. We have also reported cerebral infarction rats induce not only bladder overactivity, but also SUI. Leak point pressure measurements are the most commonly used methods to evaluate the urethral dysfunction in SUI animal models. Originally, we have developed microtransducer-tipped catheter measurements of urethral activity during sneezing. Extensive our basic research has clarified potential strategies for pharmacotherapy of SUI in the central nervous system. Therapeutic targets include adrenergic and serotonergic (5-HT) receptors in the spinal cord projected from neurons in the locus coeruleus and raphe nucleus, respectively, which stimulate pudendal nerve innervating the external urethral sphincter. Activation of α₁-adrenoceptors, 5-HT_2C, or 5-HT₇ receptors enhances the reflex at the spinal cord level whereas pre- or postsynaptic α₂-adrenoceptors and/or 5-HT_1A receptors inhibit the reflex. In addition, we have reported that stimulation of the spinal μ-opioid receptors may be a new candidate for the treatment of SUI. Thus, we review the recent advances in basic SUI research and potential targets for pharmacotherapy of SUI in the central nervous system.

Psychological stress exacerbates symptoms of bladder dysfunction including overactive bladder and bladder pain syndrome/interstitial cystitis not only in rodent models but also in human patients. Bombesin (BB)-related peptides and BB receptors in the brain have been implicated in the mediation/integration of stress responses. We have found that brain BB induces frequent urination through the BB receptors, serotoninergic nervous system and corticotropin-releasing factor (CRF) receptors. Interestingly, the BB-induced response is independent of the BB-induced activation of the sympatho-adrenomedullary outflow, one of the components of the primary systems for maintaining or reinstating homeostasis during stress exposure. These findings indicate that brain BB, 5-HT and CRF receptors could be new therapeutic targets for bladder dysfunction exacerbated by stress exposure. There are several concepts regarding central regulation mechanisms for the bladder function in the normal and pathological conditions. However, the exact brain pathophysiological mechanisms underlying stress-induced effects on the bladder are largely unknown. Therefore, our findings could pioneer a novel neuropharmacological field, central "Neuro-Uro-Pharmacology".

Iron is an essential trace metal element, and it is involved in hemoglobin synthesis, redox reaction, enzyme activity, cell proliferation and apoptosis in various cells. Therefore, iron overload has not attracted attention as compared with iron deficiency typified by anemia. Excessive iron produces hydroxyl radicals with strong oxidizing power via Fenton reaction, causing organ damage in hereditary iron overload diseases. Recent years, it has been clarified that iron accumulation is involved in the pathological conditions even in diseases including cardio-renal vascular diseases and metabolic diseases thought to be unrelated to iron so far. Therefore, the role of iron in the living body has been raised attention again.

We have revealed that iron reduction by iron chelator ameliorates adipose oxidative stress, contributing to the reduction of fat hypertrophy, macrophage infiltration and inflammatory cytokine expression in obese and diabetic KKAy mice. Dietary iron restriction also diminishes renal oxidative stress, leading to the inhibition of albuminuria excretion, glomerular lesions and inflammatory cytokines in db/db mice, a model of diabetic kidney disease.

In the presentation, we'd like to outline the role of iron on obesity diabetes and its complication, and the possibility of application to treatment with iron regulation in those disorders.

Iron is the most abundant transition metal in our body and plays various pivotal roles in our lives including oxygen transport, energy production, and metabolic reactions. At the same time, an excess amount of iron may cause cellular damages through undesired oxidative reactions. We have developed several fluorescent probes, which can detect Fe(II) ion selectively with fluorescence enhancement, to understand both the physiological and pathological contributions of iron in living systems. These fluorescent probes worked in an aqueous buffer, living cells, and histochemical samples (Chem. Sci. 2013, 4, 1250; Chem. Sci. 2017, 8, 4858; Free Radic. Res. 2014, 48, 990; Sci. Rep. 2017, 7, 10621) I will present the recent progress of the development of organelle-targeted fluorescent probes for mitochondria, lysosome, and plasma-membrane (Metallomics, 2018, 10, 794; Metallomics, 2018, in press; ACS Chem. Biol., 2018, 13, 1853). Besides, our recent approach to discover iron-regulating drug by high-throughput screening with a highly sensitive fluorescent probe will be presented.

Zinc is an essential trace element that is required for a variety of cellular functions, and unbalanced zinc homeostasis results in health problems. Recent studies have highlighted that zinc acts as a signaling mediator: zinc signal, which is controlled via zinc transporters, and participates in health and disease conditions (1). In this symposium, I will address the updated information about the roles of zinc homeostasis and zinc signaling in physiology and pathophysiology.

The first manifestations that appear under zinc deficiency are skin defects (2). It should be also noted that about 60% of whole zinc in body is kept in skeletal muscle. In addition, attentions have been drawn to zinc accumulation in tumors. However, the physiological and pathophysiological relevance, and their zinc-related molecular mechanisms underlying normal skin and muscle development, as well as the mechanism by which disturbed zinc homeostasis causes disorders including cancers, have not been clarified yet.

In this symposium, I will provide an overview of the relationships between zinc dysregulation and skin disorders, by focusing on the roles of zinc transporter ZIP7 and ZIP10 in skin formation (3, 4), and cancer cachexia mediated by ZIP14 (5). I also address the zinc homeostatic system contributes skeletal muscle formation and function via zinc signaling mediated by ZIP13.

References:

1: International Journal of Medical Sciences 18: 2708, 2017

2: Nutrients 10: 219, 2018

3: Journal of Investigative Dermatology 137: 1682-1691, 2017

4: Proc. Natl. Acad. Sci. USA 114:12243-12248, 2017

5: Nature Medicine 24:770-781, 2018

Down syndrome (DS) with an additional copy of the human chr.21 (HSA21) is characterized by various phenotypes, such as intellectual disability and neurological abnormalities. Although it is widely accepted that a high level of oxidative stress (OS) is involved in DS symptoms, the actual role of elevated OS on DS phenotypes remains unclear. Among the genes on HSA21, amyloid-β precursor protein (App) and Cu/Zn superoxide dismutase (Sod1) are thought to be involved in the elevated OS and neurological abnormalities commonly described in DS. Ts1Cje mice, a widely used genetic model of DS, exhibit some of the abnormalities of DS patients, including cognitive impairment with electrophysiological abnormality, despite the exclusion of App and Sod1 in their trisomic region. We demonstrated a high level of OS in Ts1Cje mice, suggesting that the trisomic gene(s) other than App and Sod1 increase the OS in Ts1Cje mice. In this symposium, I intend to introduce our latest results, which suggest the accumulation of copper in the Ts1Cje mouse brain, and discuss the pathophysiological significance of copper in relation to the abnormalities observed in DS patients, particularly elevated OS.

TRPV4 is a nonselective cation channel and involved in physical sensing in various types of tissues. The present study investigated the roles of TRPV4 in the pathogenesis of colitis and colitis-associated cancer in mice. The severity of colitis induced by daily treatment with dextran sulfate sodium (DSS) was significantly attenuated in TRPV4-deficient mice when compared with wild-type (WT) mice. The up-regulation of endothelial TRPV4 in colitis models suggests that this channel plays a crucial role in intestinal inflammation via increasing vascular permeability. TRPV4 activation decreased the major endothelial adhesion molecule VE-cadherin in the mouse colon and mouse aortic endothelial cells. The formation of colonic cancer induced by azoxymethane injection following DSS-treatment was also significantly attenuated in TRPV4-deficient mice. TRPV4 was co-localized with f angiogenesis marker and macrophages marker. Azoxymethane/DSS-treatment upregulated the expression of CD105 and VEGFR2 as well as TRPV4 in WT, but these responses were significantly attenuated in TRPV4-deficient mice. Activation of TRPV4 increased TNF-α and CXCL2 expression in peritoneal macrophages. These results suggest that TRPV4 expressed in vascular endothelia and macrophages contribute to progression of colitis and colitis associated cancer.

Ca_v3.2 T-type Ca²⁺ channels regulate neuronal excitability, contributing to pain signaling, and their function is enhanced by H₂S, a gasotransmitter. Ca_v3.2 is also expressed in certain cancer cells, and may affect cancer patients' prognosis. We have been studying the role of Ca_v3.2 in human prostate cancer LNCaP cells, particularly during neuroendocrine (NE)-like differentiation, which might be involved in hormone therapy resistance of prostate cancer. Ca_v3.2 and cystathionine γ-lyase (CSE), an H₂S-generating enzyme, are overexpressed in LNCaP cells during NE differentiation, accompanied by upregulation of Egr-1 and downregulation of REST, which contribute to transcriptional upregulation of Ca_v3.2. These events may participate in increased secretion of mitogenic factors essential for proliferation of surrounding cells. Further, increased extracellular glucose levels accelerate functional upregulation and overexpression of Ca_v3.2 probably through asparagine-linked glycosylation of Ca_v3.2. Our study thus suggests a crucial role of the H₂S/Ca_v3.2 system in NE-like differentiated LNCaP cells, which is promoted by hyperglycemia, being consistent with the clinical evidence that diabetes is a risk factor for castration-resistance of prostate cancer.

Cardiac glycosides such as digitoxin and digoxin are Na,K-ATPase inhibitors, and they have been used clinically for treatment of heart failure. Interestingly, lower recurrence of cancer and improvement of their survival are suggested in cancer patients who have been taking cardiac glycosides. In the cancer cells, it has been reported that sub-micromolar cardiac glycosides showed anti-cancer effects without affecting Na,,K-ATPase activity. We found recently that the receptor-type Na,K-ATPase, which has no pumping activity, is specifically associated with LRRC8A, an component of volume-regulated anion channel (VRAC), in the membrane microdomains of plasma membrane of cancer cells, and that functional relation between them is involved in the inhibitory mechanism of submicromolar cardiac glycosides for cancer cell growth. In this mechanism, binding of cardiac glycosides to the receptor-type Na,K-ATPase stimulates the production of reactive oxygen species by NADPH oxidase, and they activate VRAC within membrane microdomains, thus eliciting anti-proliferative effects. Of note, digoxin and digitoxin showed anti-proliferative effects in cancer cells at their therapeutic concentration ranges. These effects were not observed in non-cancer cells.

In cancer cells, Ca²⁺-activated K⁺ channels K_Ca1.1 and K_Ca3.1 are co-localized with Ca²⁺-permeable Orai/TRP channels to provide a positive-feedback loop for Ca²⁺ entry. They are responsible for the promotion of cell growth and metastasis in the different types of cancer, and are therefore potential therapeutic targets and biomarkers for cancer. We determined the epigenetic and post-transcriptional dysregulation of K_Ca3.1 by class I histone deacetylase (HDAC) inhibitors in breast and prostate cancer cells. We further determined the transcriptional repression and protein degradation of K_Ca1.1 by vitamin D receptor (VDR) agonists and androgen receptor (AR) antagonists, which are expected as potential therapeutic drugs for triple-negative breast cancer. A class III HDAC, SIRT1 is involved in cancer growth, stemness, and metastasis. We here introduce SIRT1-mediated regulation of K_Ca3.1 expression in breast, prostate, and colorectal cancer by three-dimensional spheroid culture. The anti-inflammatory cytokine, interleukin-10 (IL-10) is an immunosuppressive factor involved in tumorigenesis, and plays a crucial role in escape from tumor immune surveillance. We determined K_Ca3.1 activators are a possible therapeutic option to suppress the tumor-promoting activities of IL-10. These results may provide new insights into cancer treatment focused on Ca²⁺-activated K⁺ channels.

Type 2 ryanodine receptor (RyR2) is the Ca²⁺ release channel on the ER and plays a pivotal role in EC-coupling in the heart. Abnormal activation of RyR2 has been implicated in arrhythmogenic diseases. For example, in heart failure, chronic phosphorylation of RyR2 can contribute to enhanced Ca²⁺ leak from ER. In addition, mutations in RyR2 are reported to cause arrhythmogenic diseases such as catecholaminergic polymorphic ventricular tachycardia (CPVT) and long QT syndrome (LQTS). In most cases spontaneous Ca²⁺ release from ER via activated RyR2 is thought to trigger arrhythmia. We have recently established a procedure for functional evaluation of the arrhythmogenic RyR2 mutations using HEK293 expression system. Furthermore, we identified several compounds that suppress RyR2 activity via high-throughput screening using the HEK293 system. Because some of them suppressed abnormal Ca²⁺ signals in mouse cardiomyocytes, RyR2 inhibitors may be promising as novel anti-arrhythmic drugs.

Pulmonary arterial hypertension (PAH) is classified as group 1 of pulmonary hypertension. PAH is a progressive and fatal disease of the pulmonary artery. The major pathogenesis of PAH is sustained vasoconstriction and vascular remodeling of the pulmonary artery. These pathogeneses cause progressive elevations in pulmonary vascular resistance and pulmonary arterial pressure (PAP) in PAH patients. Elevated PAP leads to right heart failure and finally death. A central aspect of pulmonary vascular remodeling is medial hypertrophy, which is caused by the enhanced proliferation and reduced apoptosis of pulmonary arterial smooth muscle cells (PASMCs). Excitable abnormality in the pulmonary artery of PAH patients are mostly mediated by an elevated cytosolic [Ca²⁺]. Enhanced Ca²⁺ signaling have been reported in PASMCs from PAH patients. PASMCs express several Ca²⁺-permeable channels including voltage-dependent Ca²⁺ channels, receptor-operated Ca²⁺ channels, and store-operated Ca²⁺ channels. The expression levels of these Ca²⁺ channels are increased in the lung tissues and PASMCs of PAH patients. Targeting these Ca²⁺ channels in PASMCs may help develop novel therapeutic approach for PAH.

The polyamines, a functional foods material are considered to be essential growth factors in virtually all cells. The proposed roles of polyamines are the functioning of ion channels, nucleic acid packaging, signal transduction, cell proliferation, and differentiation, as well as regulation of gene expression. In skeletal muscle, regulation of polyamine levels is associated with muscle hypertrophy and atrophy, yet the underlying mechanisms of polyamine actions are not well defined. Here, we studied how polyamines may affect the proliferation and/or differentiation of murine myoblast progenitor C2C12 cell line. Upon polyamine treatment of C2C12 cells during induction of myogenic differentiation, the number of myotubes significantly increased. Morphologically, polyamine-treated C2C12 cells exhibited elongated cell body and became multi-nucleated myotubes. On the other hand, the polyamine did not have influence on myoblasts proliferation. Furthermore, compensatory muscle hypertrophy of C57BL6 mice underwent sciatic nerve transection of the left hindlimb was enhanced by administration of polyamines. Therefore, our study demonstrates that polyamines may play an important role in skeletal muscle homeostasis by enhancing myogenic differentiation.

Formation of skeletal muscle occurs at the stage of embryonic development as well as growth and regeneration in the adult. A skeletal muscle cell forms an elongated fiber containing multiple nuclei that are originated by fusion of precursor myoblasts. Recently, skeletal muscle-specific fusogenic transmembrane proteins Minion and Myomaker were identified and the mice lacking these factors showed severe muscle loss phenotype. In this study, we aimed to investigate the regulatory mechanism of the fusogenic proteins. C2C12 myoblast cell line was used as a model for in vitro myogenesis. The cells became elongated then fused to form multinucleated myotubes within 72 hours after induction of differentiation. By challenging kinase inhibitors previously known to affect muscle development, we found that activity of p38 MAPK but not ERK, JNK, ERK5 is required for induction of both Minion and Myomaker. Actually, the activity of p38 MAPK was continuously increased during C2C12 differentiation, and application of p38 inhibitor abrogated cell-cell fusion. Knockout of minion gene in C2C12 abrogated efficient fusion, but these cells were capable of inducing Tn-T, suggesting that myoblast fusion can be regulated independently of its differentiation. As p38 MAPK is a key mediator of stress and inflammatory signaling, it is possible that p38 mediated fusion process contributes to muscle regeneration after injury or might be involved in the pathology of abnormal muscle loss with increased inflammatory cytokines in cachexia.

Candida albicans infection can cause skin, vulvar, or oral pain. Despite the obvious algesic activity of C. albicans, the molecular mechanisms of fungal nociception remain largely unknown. Here we show that the C. albicans-specific signaling pathway led to severe mechanical allodynia. We discovered that C. albicans-derived β-glucan stimulated nociceptors depending on Dectin-1, and two pathways in inflammatory pain. The major pathway operates via the Dectin-1-mediated ATP-P2X₃/P2X_2/3 axis through intercellular relationships between keratinocytes and primary sensory neurons, which depends on the ATP transporter vesicular nucleotide transporter (VNUT). The other pathway operates via the Dectin-1-mediated PLC-TRPV1/TRPA1 axis in primary sensory neurons. Intriguingly, C. albicans-derived β-glucan has the ability to enhance histamine-independent pruritus, and VNUT inhibitor clodronate can be used to treat unpleasant feelings induced by β-glucan. Collectively, this is the first report to indicate that Dectin-1 and VNUT mediated innate sensory mechanisms that detect fungal infection.

A growing body of evidence suggests that intractable pain is associated with a decreased survival rate with a reduced quality of life in cancer patients. Recently, early palliative care has been shown to dramatically improve the survival rate of cancer patients. The tumor microenvironment refers to the cellular environment in which the tumor exists, including surrounding blood vessels, immune cells, fibroblasts and sensory neurons. Among those, the vascular and nervous systems share some critical guidance molecules. These comprehensive functions may ultimately affect tumor aggravation. It is of interest to note that the release of neurotransmitters including many of growth factors from sensory nerves could lead to neurogenic inflammation and may result in tumor progression. Therefore, we hypothesize that the activation of sensory nerves may contribute to tumor aggravation via the tumor microenvironment. In this session, we will introduce recent critical findings that could prove this hypothesis and discuss the possible mechanism of tumor growth associated with pain.

Astrocytes are critical regulators of CNS function. It has recently been proposed that astrocytes are a heterogeneous population in the developing CNS. However, whether there are regionally and functionally distinct populations of astrocytes in adulthood and if so, whether they play a role in behavior are unknown. Here we identify a population of astrocytes located in superficial lamina in the adult spinal dorsal horn (supSDH). In vivo imaging revealed that supSDH astrocytes increased intracellular Ca²⁺ levels following noxious stimulation such as by capsaicin. To examine the role of astrocytic Ca²⁺ increases in somatosensory behavior, we chemogenetically increased Ca²⁺ in supSDH astrocytes produce pain hypersensitivity to light mechanical stimulation. However, such astrogliogenic hypersensitivity was not induced by stimulating astrocytes located in SDH deeper lamina. Moreover, mechanical hypersensitivity following intraplantar capsaicin was prevented by lacking IP₃R2, an IP₃R subtype that critically contributes to astrocytic Ca²⁺ responses. Our findings identify a regionally restricted astrocyte population in the supSDH that powerfully modulates neuronal processing of mechanical information.

Diagnosis and treatment of arrhythmia are not sufficiently supported by present medical care. Therefore, we studied the applicability of human embryonic (ESC) or induced pluripotent stem cells (iPSC) to the diagnosis and treatment of arrhythmia. In the study on the diagnosis of arrhythmia, we established disease-specific iPSCs from a patient with long QT syndrome type 1 (LQTS1) and confirmed electrophysiological properties of LQTS1 after the cardiac differentiation. Computer simulation of the sympathetic activation state revealed the occurrence of early afterdepolarizations, the cause of sudden cardiac death in LQTS. In the study on the treatment of arrhythmia, we selectively obtained pacemaker-, Purkinje-, ventricular myocyte-like cells from cells differentiated from human ESCs/iPSCs using HCN4 and Mlc2v genes as labeling genes. When implanted to a rat model with atrioventricular block, the pacemaker-like cells successfully functioned as a pacemaker with an ability to respond to sympathetic stimulation. In conclusion, the application of human ESCs/iPSCs to the diagnosis of arrhythmia is promising and that to the treatment of arrhythmia is feasible.

In neuronal diseases, it is difficult to provide the satisfactory outcome only by stem cell transplantation because their pathogenesis is complex. Therefore, it should be developed a novel therapy considering the pathophysiology. We focused on migration of bone marrow-derived cell (BMDC)s to pathological lesion step by step with disease progression. We devised to apply this phenomenon, namely cell migration and targeting, to therapeutic strategy.

As application of cell migration, bone marrow transplantation was performed for the treatment of ALS mice. BMDCs migrated to the spinal cord and delayed disease progression, and this effect was enhanced by stem cell factor. Furthermore, we show other strategies with mesenchymal stem cells expressing growth factors transduced by human artificial chromosome vectors and with BMDCs as gene delivery carrier to target tissues.

As application of cell targeting, we have identified tissue specific peptides for dorsal root ganglion, microglia or astrocytes. And their peptides were incorporated into viral vectors or the complexes with therapeutic oligonucleotides to develop a novel therapy.

The combination of cell migration and targeting is a very useful tool for novel molecular therapy of neuronal diseases. This strategy is expected to have high therapeutic potential because therapeutic genes are targeted to specific cells and rescue cells gradually accumulate in pathological lesion as much as diseases progress.

Cell therapy is one of desired method for treating intractable muscular diseases, such as Duchenne muscular dystrophy (DMD). Here, we demonstrated the effective stepwise differentiation method from human iPSCs to engraftable muscle stem cells without transgene induction. We induced myotome-like population that is identified as Myf5 positive cells, which showed highly myogenic differentiation potential in vitro. Gene expression profile of purified Myf5+ cells demonstrated that the expression of Pax7 was significantly increased in Myf5+ cells at the late stage of differentiation. To assess the regeneration potential, we transplanted the Myf5+ cells at the late stage of differentiation into immunodeficient DMD-model mice. The Myf5+ cells could be engrafted in more than one hundred of host myofibers and regenerate the diseased muscles with producing dystrophin. Finally, we confirmed the recovery of muscle function after transplantation. Taken together, we demonstrate that the transplantation of the human iPSC-derived muscle stem cells with step-wise differentiation can be effective for DMD with amelioration of muscle function.

The differentiation of induced pluripotent stem cells (iPSCs), which have unlimited self-renewal capability and the potential to differentiate into any cell type in the body, provides promising cell sources for regenerative medicine. The somatic cell types differentiated from these stem cells have the potential for clinical applications, including cell therapy and drug screening. In this symposium, I will briefly summarize our novel therapeutic strategy for kidney and endocrine diseases using iPS cell technology.

Firstly, I will present our recent findings about cell therapy for renal anemia. The production of erythropoietin (EPO), a principal hormone for the hematopoietic system, by the kidneys is reduced in patients with chronic kidney disease (CKD), eventually resulting in severe anemia. Although recombinant human EPO treatment improves anemia in patients with CKD, returning to full red blood cell production without fluctuations does not always occur. Although vigorous efforts have been made to generate multiple somatic cell types from stem cells, the directed differentiation of EPO-producing cells (EPO cells) from iPSCs has not yet been achieved. Recently, we established a method to generate EPO-producing cells from human iPSCs (hiPSCs) by modifying previously reported hepatic differentiation protocols. These cells showed increased EPO expression and secretion in response to low oxygen conditions. The EPO protein secreted from hiPSC-derived EPO-producing (hiPSC-EPO) cells induced the erythropoietic differentiation of human umbilical cord blood progenitor cells in vitro. Furthermore, transplantation of hiPSC-EPO cells into mice with CKD induced by adenine treatment improved renal anemia. Thus, hiPSC-EPO cells may be a useful tool for clarifying the mechanisms of EPO production and may be useful as a therapeutic strategy for treating renal anemia.

Secondly, I will mention about drug screening and evaluation for renal anemia. It has been reported that EPO production is regulated by oxygen concentrations through hypoxia-inducible factors and their regulators, prolyl hydroxylase domain-containing enzymes (PHDs). Several PHD inhibitors are currently in clinical trials for treatment of renal anemia. Interestingly, our recent findings showed that a PHD inhibitor augmented EPO production only in hiPSC-EPO cells, but not HepG2 cells, which are an immortalized human hepatoma cell line and are widely used to investigate EPO production.  These findings suggest that hiPSC-EPO cells may provide a good model for screening PHD inhibitors for their effects on renal anemia.

Finally, I will brief summarize recent topics about cell therapy for endocrine disease using iPS cell technology.

The 3D networks of cytoskeletons are mechanistic bases for cell and tissue function (e.g. epidermal barrier function). To examine the details of the 3D networks, we extended multi-target super-resolution microscopy IRIS (Kiuchi et al., Nat. Methods 12: 743-746, 2015) to 3D imaging. IRIS uses exchangeable probes that directly associate with and dissociate from their targets. By integrating single-molecule localization and sequential labeling, IRIS enables the high resolution imaging of multiple targets within a single specimen. By the repeated associations of the probes, the loss of fluorescent signals due to photobleaching during the 3D imaging can be compensated. Moreover the labeling density on the target structures can be unlimitedly increased, yielding greatly improved continuous labeling along the cytoskeletal filaments. We developed IRIS probes for three cytoskeletons and focal adhesions from the target-associated proteins. For 3D IRIS imaging, we combined the IRIS method with HILO illumination and astigmatism using adaptive optics. This allowed us to discern microtubules at heights of 6.3 μm and 6.5 μm in the apical region of the cell. Thus IRIS is a potent tool to monitor the 3D network of cytoskeletons.

Multicellular organisms including human are constituted of cells with different function and identity. Cell identity depends on gene expression programs. Single nucleotide polymorphisms in super-enhancers have recently been associated with a wide range of human diseases (Hnisz et al., 2013, Cell, 155, 934-47), suggesting the destabilization of cell identity causes disease. However, the mechanisms that suppress cell-type gene expression programs are poorly understood. Here we show that serum response factor (Srf), a transcription factor that is activated by various extracellular stimuli such as extracellular matrix stiffness, can suppress cell-type gene expressions. Depletion of β-actin increased the nuclear localization of Mkl1, a cofactor of Srf, resulting in the activation of Srf. Misactivation of Srf, which can be achieved by the suppression of Actb (β-actin-encoding gene) as well as overexpression of Mkl1 or Srf downregulated cell-type gene expressions. Transgenic mice overexpressing Srf exhibited various symptoms associated with cell identity loss. Moreover, analyses of publicly available microarray data of human diseases suggested that the misactivation of Srf is also associated with various human diseases. Srf could be a possible target molecule for therapy of various diseases.

Non-syndromic HSNHL occurs 1 in 1000 live births. Newborn hearing screening had been introduced around 2000; however, no effective therapy for SNHL has been developed. About 100 genes associated with non-syndromic HSNHL has been discovered: about 30 of these encode proteins associate with actin. We are intensively studying about Rho-family GTPases, which are key regulators for actin structures. During the study of hair cell-specific Cdc42-KO mice, we found that the activated RhoA signaling is one of causes of progressive SNHL in Cdc42-KO mice. RhoA is a regulator of DIA1, which is a key molecule in straight actin elongation and the responsible gene of the 1st type of autosomal dominant non-syndromic SNHL, DFNA1. We have discovered novel DFNA1 patients and clarified that DFNA1 is caused from constitutively active mutants of DIA1, in which the auto-inhibitory interaction is disrupted. Besides, patients with CDC42 mutants showing HSNHL were reported from Japan in 2015, and they are categorized into Takenouchi-Kosaki syndrome.

I will present recent advances of our studies regarding SNHL and our trial for development of novel drugs against SNHL.