Insulin-derived amyloidosis is a skin-related complication of insulin therapy and is caused by the insulin amyloid deposits at the insulin injection sites. Insulin-derived amyloidosis causes poor glycemic control and increased insulin dose requirements because of impaired insulin absorption at the amyloid sites. There are two forms of insulin-derived amyloidosis, with and without palpable masses. Insulin-derived amyloidosis with a palpable mass may often be called "insulin ball". Lipohypertrophy is another skin-related complication of insulin therapy and is common in patients on insulin treatment. Insulin-derived amyloidosis and lipohypertrophy are often confused because they have similar characteristics and may coexist. Since insulin-derived amyloidosis and lipohypertrophy interfere with insulin therapy, it is important to prevent them. The greatest risk factor for insulin-derived amyloidosis and lipohypertrophy is the repeated insulin injections at the same site. Therefore, it is necessary to regularly observe the insulin injection sites and repeatedly educate the correct insulin injection methods. For this purpose, not only physicians but also medical staff need to cooperate.

Recently, in the field of diabetes, it has been noticed that subcutaneous injection to the insulin ball can cause poor blood glucose control. Therefore, it is important to prevent insulin ball.

" The Japanese Pharmacological Society will conduct more educational activities on pharmacological knowledge necessary for nursing through symposia and seminars starting in 2018, and also aim to bridge human interaction between pharmacology and various fields of nursing that have not had much interaction with pharmacology." (from the Japanese Pharmacological Society website). With this aim in mind, it was decided that "Scoping Review: Insulin Ball" be conducted as an activity for the joint conference project.　

Therefore, this scoping review was conducted to address the research questions, "What methods are used to assess insulin injection sites?" and "What is the preventive care for induration and how effective is it?"  In most of the literature, the injection site was confirmed by palpation and visual examination, and there was also some literature that incorporated ultrasound. There were many reports of insulin balls occurring because patients have been injecting insulin at the same site, even though healthcare professionals instructed them to avoid the same site. Some of the literature had specific teaching methods such as hand site rotation and calendar injection method.

Recently, FDA approved psilocybin, the psychoactive substance found in the magic mushroom, as a "breakthrough therapy" for depression; however, its detailed mechanism remains unknown. Serotonergic psychedelics such as psilocybin and LSD have hallucinatory effect through stimulation of serotonin 5-HT_2A receptor (5-HT2A), motivating us to investigate the role of 5-HT2A stimulation in antidepressant effect of serotonergic psychedelics as well as its neural basis in mice. We demonstrated that 5-HT2A agonists such as DOI and psilocin, an active metabolite of psilocybin, decreased immobility time in the forced-swim test (FST), which was absent in mice with knockdown of Htr2a (5-HT2A gene) in the lateral septum (LS) by AAV-delivered shRNA. Since 5-HT2A is a Gq-coupled GPCR, we next investigated the effect of Gq signaling activation in 5-HT2A-positive neurons in LS on emotional behaviors in mice, for which hM3Dq, a Gq-coupled designer receptor activated by CNO, was transfected into 5-HT2A-positive neurons in LS of Htr2a-cre mice by microinjection of cre-dependent AAV. Activation of the hM3Dq with CNO induced antidepressant and anxiolytic effects in mice. Further, we found that most of 5-HT2A-positive cells in LS were the GABAergic inhibitory neurons, suggesting that activation of 5-HT2A-positive GABAergic neurons in LS leads to antidepressant effect. In this symposium, I would like to introduce the role of 5-HT2A in LS in not only antidepressant effect but also hallucination of serotonergic psychedelics.

Tryptophan (TRP) is metabolized via the kynurenine (KYN) pathway, which is related to the pathogenesis of major depressive disorder (MDD). Kynurenine 3-monooxygenase (KMO) is a pivotal enzyme in the metabolism of KYN to 3-hydroxykynurenine. In rodents, KMO deficiency induces a depression-like behavior and increases the levels of kynurenic acid (KA), a KYN metabolite formed by kynurenine aminotransferases (KATs). We will introduce the involvement of TRP metabolism in the depression-like behavior induced by chronic unpredictable mild stress (CUMS). Corticosterone level in the serum and corticosterone-releasing hormone (CRH) mRNA level in the hypothalamus (HT) were elevated immediately after CUMS. Associated with the dysregulation of hypothalamic-pituitary-adrenal (HPA) axis, KMO mRNA level was decreased, and KA content was increased in the prefrontal cortex (PFC). Microglia marker Iba-1 was decreased immediately after CUMS. Because KMO is mainly found in microglia in the central nervous system, these results suggests that CUMS increased KA contents via alternation of kynurenine metabolic enzyme from KMO to KATs due to the reduction of microglia. Because KA is α7 nicotinic acetylcholine receptor (α7nAChR) antagonist, we investigated the effect of nicotine and galantamine (allosteric potentiating ligand for α7nAChR ) on the depression-like behavior and dysregulation of HPA axis induced by CUMS. When nicotine and galantamine were administrated before exposure to each stressor, they attenuated CUMS-induced depression-like behaviors. Although nicotine didn't affect elevated corticosterone level in the serum immediately after CUMS, it suppressed the sustained elevation 1 week after CUMS. Increase of KA associative with downregulation of KMO in microglia was involved in the depressive-like behavior and the sustained elevation of serum corticosterone after CUMS. The ameliorating effects of nicotine and galantamine on depression-like behaviors induced by CUMS are associated with the activation of α7nAChR.

Literature has shown that peroxisome proliferator-activated receptor α (PPARα), a ligand-activated transcription factor, regulates fatty acid metabolism. Our previous study has suggested that PPARα plays a crucial role in the pathophysiology of schizophrenia (Maekawa et al., 2017). In the current study, we screened and identified rare variants in the PPARA gene (encoding PPARα) of schizophrenia subjects. In vitro study showed that those variants decreased activities of PPARα as a transcription factor. Ppara KO mice exhibited a deficit in the sensorimotor gating function and schizophrenia-related histological abnormalities. RNA-seq analysis revealed that PPARα regulates the expression of synaptogenesis signaling pathway-related genes in the brain. Remarkably, treatment of inbred mice with the PPARα agonist fenofibrate alleviated an NMDA receptor antagonist, phencyclidine (PCP)-induced spine pathology and reduced sensitivity to MK-801, another NMDA receptor antagonist. In conclusion, the current study further supports the idea that perturbation in the PPARα-regulated transcriptional machinery leads to a predisposition to schizophrenia, probably affecting synapse physiology. This study also demonstrates that PPARα can serve as a novel therapeutic target for schizophrenia.

Schizophrenia is a complex multifactorial disease with typically unmet medical need. Vasoactive intestinal peptide receptor 2 (VIPR2), also known as VPAC2, is a seven transmembrane heterotrimeric G protein-coupled receptor (GPCR) that binds two homologous neuropeptides with high affinity, vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP), and it has been considered an attractive drug target for development to treat schizophrenia. However, VIPR2 belongs to class-B GPCRs that are intractable targets for small molecules. We recently developed a novel peptide KS-133 with a relatively low molecular weight (about 1500 g/mol) and characteristic bicyclic structure. KS-133 possesses drug-like properties as follows: (a) high selectivity and a potent antagonist activity against VIPR2; (b) remarkable resistant to protease degradation, a common problem with peptides; (c) prevention of cognitive decline in a mouse model of psychiatric disorders. Our study is not only the first validation of the effects of a VIPR2 antagonist in disease animal models, but also a good example of drug discovery for class-B GPCRs.

In neurodegenerative diseases such as Alzheimer's disease (AD), the appearance and accumulation of aggregates of Amyloid beta (Aβ) and phosphorylated Tau is triggered by some stimulus such as an immune response. Inflammatory bowel disease was also reported to be a risk factor for dementia in humans, but the mechanism remains unclear. The aim of this study is to develop novel therapeutic strategies by clarifying the immune responses that regulate the expansion of cells expressing Aβ and phosphorylated Tau.

We found that colitis enhanced the pathogenesis of AD, and immune cells were infiltrated in the brains and dura maters of mice models of AD by using scRNAseq, suggesting the involvement of immune responses in the development of neurological diseases.

Macrophages have been discovered more than 100 years ago. Recent studies indicated that monocytes and macrophages can be categorized into several distinct phenotypes and their respective differentiation mechanisms are known. We also reported that the Jmjd3 is critical for the macrophage subtype activated by allergic stimuli (Nat. Immunol. 2010) and that the tissue resident macrophage subtype in adipose tissue, which is controlled by Trib1, is responsible for maintaining homeostasis of peripheral tissues such as adipocyte (Nature. 2013). Thus, it is considered that various macrophage/monocyte subtypes corresponding to certain disorders were existed in our body.

Furthermore, in order to investigate the relationship between macrophage subtype and disease, we focused on fibrosis as the next target disease. Its pathogenesis is poorly understood, and there are few effective therapies. Previously we found that a new macrophage/monocyte subtype, which their markers are Msr1⁺Ceacam1⁺Ly6C^–Mac1⁺F4/80^–monocyte and share granulocyte characteristics, involved in development of fibrosis was accumulated in the affected area in the lungs at the beginning of fibrosis. We termed the monocyte/ macrophage subtype segregated-nucleus-containing atypical monocytes (SatM) (Nature. 2017). Towards understanding the mechanism of fibrosis onset, we next focused on investigation of non-haematopoietic cells involved in activation of immune cell such as SatM during fibrotic phase.

We have been studying the mechanism of myeloid cell development from the viewpoint of chromatin regulation by transcription factors. The transcription factor IRF8 is essential for the development of monocytes and dendritic cells (DCs), whereas it inhibits neutrophilic differentiation. We have demonstrated that IRF8 establishes enhancer landscapes in myeloid progenitors to epigenetically prime these cells to differentiate into monocytes or DCs. Recently, we focus on high-order chromatin structure during DC development. Our Hi-C data revealed that DC-specific active compartments are gradually established throughout the course of differentiation, while most of the topologically associating domains (TADs) specific for the DC lineage are formed with slower kinetics. We also found that the activation of DC-specific enhancers proceeds the compartment switch and subsequent gene expression. In addition, our data suggest that IRF8 is required for many of the DC-specific changes from compartment B to A. Collectively, myeloid cell gene expression patterns are epigenetically regulated by key transcription factors such as IRF8 via enhancer establishment and chromatin structure reorganization.

The central nervous system (CNS) host a variety of immune cells including macrophages, which are found either in the parenchyma or at the CNS interfaces, such as meninges and perivascular spaces, where they carry out various functions during not only neural development and homeostasis, but in neurodegenerative and neuroinflammatory diseases. Recent emergence of single-cell technologies enhanced our understanding of their origins, fates, and functional heterogeneity of these macrophages during health and perturbation. In this symposium, I would like to discuss the current knowledge about spatio-temporal and functional heterogeneity of CNS macrophages during development, homeostasis and disease, and their potential as a novel therapeutic target for CNS diseases.

Cancer ecosystems comprise not only cancer cells but also various stromal cells, such as mesenchymal and vascular endothelial cells. Pancreatic cancer is stroma-rich cancer, and the abundant stroma is considered responsible for the resistance to treatment and for poor prognosis. Accordingly, it is important to understand and control pancreatic cancer cell-stromal cell interaction. To analyze the interaction and further accurately assess the susceptibility to pancreatic cancer drugs, a culture system capable of recapitulating pancreatic cancer ecosystem is essential. We previously established a method to generate multi-lineage functional organs using human iPS cells (Takebe T, Sekine K et al, Nature, 2013; Sekine K, Camp JG et al, Nature 2017). By applying the three-dimensional tissue reconstruction technique to pancreatic cancer research, we succeeded in reconstituting human pancreatic cancer tissue (pancreatic cancer organoid) and recapitulating abundant stroma from patient-derived primary cancer cells. Evaluation of susceptibility of pancreatic cancer organoids in vitro and in vivo revealed that sensitivity to gemcitabine, a therapeutic drug for pancreatic cancer, is greatly reduced compared to the cancer cell only. In summary, pancreatic cancer organoids are an effective tool for reproducing the treatment resistance of pancreatic cancer patients and useful for drug screening. Currently, we are working on detailed analysis of patient tissue and recapitulation of advanced tissue structure with the aim of further improving the accuracy as a patient-derived cancer model.

Cancer arises primarily through accumulation of genetic mutations. Although derivation of induced pluripotent stem cell (iPSC) does not require changes in genomic sequence, somatic cells acquire the unlimited growth potential during reprogramming, a characteristic shared with cancer cells. Indeed, our previous studies have revealed an impact of reprogramming-related epigenetic regulation on development of various cancers. We showed that transient expression of reprogramming factors in vivo results in development of cancer that resembles human pediatric cancers. Indeed, the partial reprogramming-induced kidney tumors exhibited similar characteristics with human Wilms' tumors, which include aberrant DNA hypermethylation at H19 imprinting control region (ICR). Notably, the tumor cells gave rise to iPSCs that could contribute to chimeric mice. Moreover, the chimeric mice were born and grew normally without an obvious phenotype. Here we show that in vivo reprogramming with higher levels of reprogramming factors causes development of cancers that exhibit shared characteristics with human germ cell tumors. Like germ cell tumors, propagated tumor cells could differentiate into trophoblasts. In sharp contrast to H19 ICR hypermethylation in Wilms' tumor-like cancers, these tumors display a global reduction in ICR methylation, which is an unique epigenetic aberration in human germ cell tumors. Remarkably, germ cell tumor-like cancer cells give rise to iPSCs with the expanded differentiation potential to trophoblasts, which could contribute to extra-embryonic cells in the placenta as well as non-neoplastic somatic cells in vivo. Collectively, these findings underscore the central role of epigenetic regulation, but not genetic transformation, in the development of particular types of cancer.

The use of patient-derived iPS (induced-pluripotent stem) cells has become very meaningful for the development of pathological models and therapies for neuronal diseases. However, when modeling pathological conditions in vitro, it is very important to know which cell culture method to use. There are strong and weak points to each type of stem cell-based culture. We have been developing neuronal induction methods for pathological modeling of psychiatric and developmental disorders. In this study, we used both 2-D and 3-D cultures effectively. Directed neural differentiation by Dual SMAD inhibition and addition a morphogen allows us to observe neurogenesis-based tissue formation, which may reveal cellular phenotypes during development: this is achieved by 3D culture system. However, it is not always possible to obtain disease-causing cells under these conditions. In this case, it would be useful to use transient expression of transcription factors in iPS cells to directly differentiate into subtype-specific cells: this is achieved by 2D culture system. Each differentiation and culture technique is constantly evolving. It will be necessary to consider how to generate phenotypes more robustly. In this symposium, we introduce our research on modeling of psychiatric and developmental disorders based on customized neural differentiation system.

Refractory pain disease plagues many people, and relieving chronic pain is a major challenge for all patients. Fibromyalgia, which is classified as intractable pain, is a disease that causes widespread severe pain and is known to be accompanied by various concomitant symptoms such as physical and psychiatric symptoms. On the other hand, fibromyalgia is difficult to identify on the basis of common blood tests and diagnostic imaging. Therefore, there is an urgent need to establish clear diagnostic and therapeutic methods. For the purpose of finding the functional changes of neural subtypes observed in fibromyalgia pathology, we have created the differentiation induction technology from fibromyalgia-specific iPS cells. Currently, we are working to identify the target molecules involved in fibromyalgia pathology using differentiated sensory neurons, mesencephalic dopaminergic neurons and GABAergic neurons form patient-oriented iPS cells. Our results suggest that the synchronicity of abnormal peripheral sensory responses and impaired mesencephalic dopamine neurotransmission may be involved in the development of hyperalgesia and psychotic symptoms in fibromyalgia. This symposium outlines a new approach using disease-specific iPS cell technology to elucidate the mechanisms of the development of refractory diseases such as fibromyalgia.

A variety of new methods are being tried in education of pharmacology for medical students, to make pharmacology be directly oriented to practical medical treatment. Among them, thinking that the method of "selection of personal drug (P-drug)" is suitable for learning "evidence-based medicine (EBM)", I have been engaged in "P-drug education" in Kyushu university for many years. If doctors carefully select medicines that are indispensable for their medical treatment based on clinical evidence, are made familiar with how to use them, and in principle perform daily medical treatment using only those medicines, EBM can be really practiced. And moreover, it may also lead to the suppression of medical errors and adverse drug reactions. Such essential medicines for an individual doctor are called P-drug. Since 2003, I have adopted "P-drug selection" in the education of pharmacology for upper grade medical students. After more than 15 years of trial and error, I have been able to create an educational model using "P-drug selection" that I think could be easily adopted at any medical school. At this symposium, I would like to talk about the relationship between "P-drug selection" and EBM and demonstrate the "P-drug education" model.

The active learning of practical pharmacotherapy has been performed at 22 medical schools, 2 dental schools, one Pharmacy school and one Nursing school as a part of pharmacological education from 2010 to 2021. This active learning program is named Case & Communication based approach (C&C approach), since it is studied through communication between medical doctors / dentists / pharmacists / nurses and patients based on cases presented beforehand. The educational effectiveness was evaluated by a questionnaire survey. The highly effectiveness of role-play in pharmacology education was observed regarding all four topics: (a) understanding of medical treatment, (b) understanding patient's feelings, (c) improvement of awareness and motivation as a medical stuff and (d) positive influence upon study attitude. In addition, many students realized the importance of physician-patient communication, understanding of patient's feeling, and accurate and extensive knowledge of pharmacotherapy in their free description. This role-play program in pharmacology education may be effective for better understanding of pharmacotherapy, patient's feeling, and improvement of students' awareness and motivation as a medical stuff.

The program of pharmacology role-play was started as a part of pharmacological Practice for 3rd-grade medical students at Kurume University in 2013. Pharmacology role-play is active learning program, in which medical students learn how medical doctors chose medicines to treat the presented cases and explain the medical treatment to patients. Students have the basis of pharmacotherapy after almost 1 year of pharmacology course, and this program becomes the first step of the education of clinical pharmacology. Pharmacology role-play was highly evaluated by students, as presented by other symposiasts. However, we realized that this program works well for students playing the role of doctor or patient, but that students without any roles behave like observers. To improve this issue, personal drug training was added to the program of pharmacology role-play in 2016. All the students, not only those playing the role of doctor, need to select therapeutic drugs for the presented cases based on the elements of personal drug (i.e. efficacy, safety, compatibility and cost). This process is required to make a report of personal drug, and facilitate the discussion at pharmacology role-play. Thus, the program of pharmacology role-play integrated with personal drug training seems useful to make all students participate in the activity and to improve motivation for learning clinical pharmacology. In this session, we will discuss the effectiveness of this integrated program.

The pharmacology role play started at Miyazaki University in 2010, and is being implemented as a common program at 23 schools in 4 faculties of medicine, dentistry, medicine, and nursing by 2020. It is one of the active learning of pharmacology by the Case & Communication based approach (C & C approach) because it learns based on the case presented in advance and actively learns through communication. Student questionnaire surveys conducted at each facility show that it is highly effective in understanding drug treatment, understanding patients' feelings, and improving motivation, and it is changed from "mere memory" to "living knowledge". It can be said that it is an effective active learning. However, the results so far have been obtained only within one facility, and have not been implemented among larger facilities, including multiple occupations. However, the spread of COVID-19 infection in 2020 was a turning point that drastically changed the way of medical school education centered on traditional face-to-face lectures, and as a result, various styles of distance education were implemented. Among them, real-time lessons using Zoom etc. have the advantage that about 300 students can be conducted at multiple facilities without having to gather them in one place at the same time. With the corona vortex as a strange currency, the infrastructure has been set up to carry out joint education in pharmacology role-playing among multiple facilities. This time, we conducted a pharmacology role-play jointly by Fujita Health University and Aichi Medical University. We would like to report on the experience gained from the joint implementation of the two universities.

Live-cell super-resolution microscopy is a powerful tool that reveals spatiotemporal dynamics of biomolecules with nano-scale resolution in live specimens. However, the photobleaching of fluorescent molecules labeled to the target molecule is a significant obstacle to continuously obtaining super-resolution images for a long period. In this study, we developed a fluorescent labeling technique called de-quenching of organic dye emission (DeQODE) that enables long-term observation with a super-resolution microscope without being affected by photobleaching. In the DeQODE, a non-fluorescent probe is designed to become fluorescent by sequential binding to tag protein, an antibody against the probe, enabling maintaining the fluorescence signal for a long period. After optimizing the binding-dissociating property between tag protein and the probe, several intracellular organelles and the distribution of proteins labeled with DeQODE were imaged with a nano-scale resolution for tens of minutes by multiple super-resolution microscopies including stimulated emission depletion microscopy (STED), single-molecule localization microscopy (SMLM), and structured illumination microscopy (SIM). Our results show that DeQODE can contribute to research clarifying the nano-scale dynamics of functional molecules by live-cell super-resolution imaging.

Membrane receptors including G protein-coupled receptors (GPCRs), receptor tyrosine kinases (RTKs) and ion channels are major drug targets. We have demonstrated that GPCR molecules generally accumulate into membrane domains upon activation. Here we show that the signaling bias of an angiotensin receptor (AT1R) is regulated by the membrane domain colocalization of receptor/G protein/GRK using multicolor single-molecule imaging. We also show that the different kinetics of endocytosis between TRPV1 and TRPV4 channels are derived from the pre-accumulation of TRPV channels in membrane domains before activation by single-molecule time-lapse imaging. Single-molecule imaging provides us a lot of information including protein-protein interaction, diffusion dynamics, and oligomerization of proteins in living cells, which cannot be obtained by a conventional high content analyzer. In order to utilize the single-molecule imaging to pharmacology, we are currently developing a multicolor single-molecule imaging system that enables us to monitor cells in a 96-well plate automatically. We will discuss the future development of the next generation high content analyzer based on single molecule imaging.

Two-photon excitation laser scanning fluorescence microscopy (TPLSM) is a powerful tool to visualize intravital microstructures. This is because of its superior penetration depth and less-invasiveness in specimens owing to its near-infrared excitation laser wavelength compared with the single-photon excitation. In this presentation, our studies to improve spatial and temporal resolution of TPLSM by utilizing some of novel optical technologies are introduced. To improve the spatial resolution, we applied stimulated emission depletion (STED) technologies utilizing electrically controllable components, transimissive liquid crystal devises and laser diode-based light sources [Opt. Express 2014; Biomed Opt. Express 2018], resulting in 5-times hihger spatial resolution than conventional [Biomed Opt. Express 2019]. In addition, we recently adopted other technologies such as adaptive optics [PLOS One 2020; ACS Omega 2021] or nano-sheet based observation methods [iSci. 2020; Star Protocol's. 2021] for sharper intravital imaging. For higher temporal resolution, a confocal spinning disk scanning unit and high-peak-power laser light sources were utilized for TPLSM imaging [Anal. Sci. 2015; Front. Physical. 2019; BBRC 2020]. The developed system achieved large field of view, fine spatial resolution and penetration depth, and has been used for several biological applications.

Lewy body dementias (LBD) are classified into Parkinson's disease, Lewy body dementias, and pure autonomic failure according to the main lesion, and the lesions overlap in principle.

On the other hand, we have clarified that the olfactory bulb / amygdala subtype secondary to Alzheimer's disease is characterized by the absence of Lewy bodies in the peripheral autonomic nervous system. In addition, multiple system atrophy and LBD-accumulated α-synuclein are stained with Gallyas silver stains, the former being positive and the latter being negative, and it is said that they differ in RT-QUIC and transmission to experimental animals. In LBD, cortical Lewy bodies and brainstem Lewy bodies have morphological differences with or without cores, and differences due to RT-QUIC have been reported. However, the differences in our previous Western blot studies did not confirm the differences observed in prion disease. There has been no report on the difference in the mechanism of α-synuclein aggregation in the Lewy body of the peripheral and central nervous system between primary and secondary LBD, and this is a topic for the future.

In multiple system atrophy (MSA) patients, alpha-synuclein (ɑSyn) predominantly accumulates in oligodendrocytes and forms glial cytoplasmic inclusions (GCIs). Such changes induce oligodendrocyte degeneration and demyelination, and damages trophic support of glial cells to neurons. The toxic formations of aSyn oligomers or fibrils in glial cells are triggered by cellular oxidative stress. Exposure to polyunsaturated fatty acids such as arachidonic acid (AA) increases reactive oxygen species (ROS) in neurons. Fatty acid-binding proteins (FABPs), a family of transporter proteins, bind with high affinity to long-chain fatty acid such as AA. FABP7 is primarily expressed in the oligodendrocytes and neural stem cells. To elucidate FABP7's involvement in MSA, we overexpressed ɑSyn in U251 human glioblastoma cells and confirmed FABP7 toxicity via its enhancement of ɑSyn oligomer formation. FABP7-induced ɑSyn aggregation significantly increased by exposure to AA in U251 cells. FABP7-induced ɑSyn oligomerization was associated with cell death in U251 cells. FABP7-induced ɑSyn oligomerization was also evidenced in both KG-1C human oligodendroglial cells and oligodendrocyte precursor cells (OPCs). FABP7 ligand 6 (BRI-601) disrupted the FABP7–ɑSyn interaction in these glial cells. Psychosine treatment also triggered ɑSyn oligomerization by FABP7 through phospholipase A2 activation, and induced KG-1C and OPCs cell death. BRI-601 also rescued the psychosine toxicity in glial cells. Taken together, FABP7 triggers aSyn oligomerization associated with oxidative stress, while BRI-601 can prevent the oxidative stress-induced ɑSyn toxicities, thereby rescuing MSA progression.

Liquid droplets of aggregation-prone proteins, which become hydrogels or form amyloid fibrils, are a potential target for drug discovery. In this study, we proposed an experiment-guided protocol for characterizing the design grammar of peptides that can regulate droplet formation and aggregation. The protocol essentially involves investigation of 19 amino acid additives and polymerization of the identified amino acids. As a proof of concept, we applied this protocol to fused in sarcoma (FUS) and succeeded the peptide binder design and aggregation regulation. The developed protocol could be used for the primary design of peptides controlling liquid droplets and aggregates of proteins. Also, I may introduce the development of rational peptide binder design method.

Intracellular accumulation of α-synuclein is a pathological hallmark of Lewy body diseases, including Parkinson's disease and Dementia with Lewy bodies. Accumulation of pathogenic proteins is associated with the process of uptake into neuronal cells. Various molecules participate in the α-synuclein uptake and propagation. Here, we introduce the unique process of α-synuclein uptake and show the impact of fatty acid-binding protein 3 (FABP3) with dopamine D2 receptors. FABP3 is rich in dopaminergic neurons and interacts with dopamine D2 receptors, specifically the long type (D_2L), abundant in caveolae. We demonstrated that mesencephalic neurons with tyrosine hydroxylase (TH)⁺, FABP3^-/-, D_2L^-/- do not take up α-synuclein. Likewise, dynasore, a dynamin inhibitor, and caveolin-1 knockdown also abolished the uptake. Second, we advance the possible preventives for α-synuclein propagation. We found that the deletion of α-synuclein C-terminal end abolished the uptake to dopaminergic neurons. Additionally, exposure to the C-terminal peptides alleviated the α-synuclein uptake. The antagonistic action on the α-synuclein-FABP3 binding is different from the FABP3 ligands. Based on these data, we propose a novel pathogenic mechanism of Lewy body diseases and potential therapeutic medicines targeting the α-synuclein uptake process.

Cyclin M (CNNM) constitute a family of proteins that function as Mg²⁺-extruding transporters by stimulating Na⁺/Mg²⁺ exchange, and thereby control intracellular Mg²⁺ levels. This CNNM-mediated Mg²⁺-extrusion is inhibited by the direct binding of phosphatase of regenerating liver (PRL), of which overexpression is frequently observed in malignant cancer tissues including colorectal cancer metastasis, and such inhibition is responsible for the malignant progression driven by PRL. Studies with CNNM knockout mice revealed the importance of CNNM4 and CNNM2 in maintaining organismal Mg²⁺ homeostasis by mediating intestinal Mg²⁺ absorption and renal reabsorption, respectively. Moreover, CNNM are known to be involved in various diseases; mutations in CNNM2 and CNNM4 cause dominant familial hypomagnesemia and Jalili syndrome, respectively, and genome wide association studies have also revealed the importance of CNNM2 in multiple major diseases including hypertension and schizophrenia. Collectively, the molecular and biological characterizations of CNNM family show that they are interesting therapeutic targets. The current status of drug development targeting these molecules is also discussed.

The CNNM/CorC family proteins are Mg²⁺ transporters that are widely distributed in all domains of life. In bacteria, CorC has been implicated in the survival of pathogenic microorganisms in their host environment and in resistance to antibiotic exposure. In humans, CNNM proteins are involved in a number of biological events, such as body absorption/reabsorption of Mg²⁺, hypertension, genetic disorders and tumour progression. Accordingly, both CorC and CNNM have attracted interest as therapeutic targets.

Despite the physiological importance of the CNNM/CorC family proteins, their Mg²⁺ transport mechanism is unclear, mainly due to the lack of a CNNM/CorC family protein structure that includes its transmembrane (TM) domain.

In this talk, we present the crystal structure of the CorC TM domain dimer in the presence of Mg²⁺. The CorC TM domain dimer consists of six TM helices surrounded by long, curved, amphipathic helices parallel to the membrane. Each protomer possesses a single Mg²⁺ binding site with a fully dehydrated Mg²⁺ ion coordinated to five amino acids, which highly contrasts with that of the known Mg²⁺ channel structures, where Mg²⁺ ions are typically fully-hydrated. The residues at the Mg²⁺ binding site are strictly conserved in both human CNNM2 and CNNM4, and many of these residues are associated with genetic diseases in humans.

Pulmonary arterial hypertension (PAH) is a severe and progressive disease that leads to right heart failure. The pathogenesis of PAH is generally characterized by vasoconstriction, upregulated proliferation, migration, and pulmonary vascular remodeling in lung tissue. Recent studies using genetic analyses and experimental models have suggested that the hypercontraction of pulmonary arteries induced by Ca²⁺ signaling abnormality may be involved in the pathogenesis of PAH. However, the pathological functions of Ca²⁺ transporters in PAH are not clearly understood. The Na⁺/Ca²⁺ exchanger type-1 (NCX1) is a bidirectional transporter that is controlled by membrane potential and transmembrane gradients of Na⁺ and Ca²⁺. Vascular smooth muscle NCX1 plays an important role in intracellular Ca²⁺ homeostasis and Ca²⁺ signaling. In this study, we focused on the pathological role of NCX1 in hypoxia-induced PAH model. Since the expression of NCX1 was markedly increased in the pulmonary arteries of mice exposed to chronic hypoxia, we generated hypoxia-induced PAH model using NCX1 heterozygous knockout mice as well as vascular smooth muscle specific NCX1 conditional knockout mice. Both NCX1 knockout mice exhibited significant reduction in right ventricular systolic pressure and hypertrophy, compared with wild-type mice. In addition, hypoxia-induced pulmonary vessel muscularization was also significantly attenuated in both NCX1 knockout mice. Furthermore, specific NCX1 inhibitor SEA0400 significantly suppressed hypoxia-induced PAH and pulmonary vessel muscularization, with a slight reduction in right ventricular hypertrophy in wild-type mice. These findings indicate that the upregulation of vascular smooth muscle NCX1 contributes to the development of hypoxia-induced PAH, suggesting that NCX1 inhibition might be a novel approach for the treatment of PAH.

Circadian clock generates many physiological rhythms. In mammals, most of the rhythms are generated by transcriptional rhythms based on transcriptional and translational feedback loops (TTFL). Importantly, period lengths of the rhythms are almost constant in the range of physiological temperatures, and the property is called as temperature compensation.

In order to understand the mechanism of the temperature compensation, we screened small-molecule inhibitors by using Rat-1 fibroblasts expressing luciferase reporter of the transcriptional rhythms. The temperature compensation was compromised in the presence of an inhibitor of NCX (KB-R7943, SEA0400) or CaMKII (KN-93). Further analysis revealed that temperature lowering enhances NCX-dependent Ca²⁺ influx to activate CaMKII. CaMKII facilitates heterodimerization of CLOCK and BMAL1, bHLH transcription factors, thereby activating gene expression through E-box DNA elements. Thus, NCX-dependent cold Ca²⁺-CaMKII signaling compensates for deceleration of TTFL at lower temperatures. In addition to the temperature compensation, activities of NCX and CaMKII are essential for cell-autonomous oscillation of the rhythms. These results demonstrate that NCX is a new target for drug discovery based on the circadian clock function. (Kon et al., Genes and Development, 2014; Kon et al. Science Advances, 2021).

Cognitive flexibility is required for decision-making behavior, and cognitive impairment can be observed in various psychiatric disorders. Nucleus accumbens (NAc) is one of key neural substrates for cognitive behavior in the cortico-basal ganglia circuit. Within the NAc, dopamine D1 and D2 receptor-expressing medium spiny neuron (D1-/D2-MSNs) in the direct and indirect pathways have been revealed to play important roles in controlling reward and aversive behavior, respectively. However, the collaborative role of NAc D1-/D2-MSNs in flexible cognitive behavior has been remained. In this study, using a visual discrimination task in mice, we assessed the role of NAc D1-/D2-MSNs in cue-guided reward-based decision-making behavior. Cell-type specific neuronal silencing and in vivo calcium imaging revealed NAc D1-/D2-MSNs to separately contribute to cue-guided reward-based decision-making behavior. Our findings indicate that homeostatic regulation of neural circuit mechanism within NAc underlies flexible cognitive behavior and the pathophysiology of psychiatric disorders.

The spatiotemporal organization of neuronal firing is crucial for information processing, but how thousands of synaptic inputs to the dendritic spines drive the firing remains a central question in neuroscience. Although a change in synaptic density and strength have been a responsible factor for the pathophysiology of various psychiatric disorders, it is entirely unclear what changes in neuronal computation and subsequent dynamism of neuronal circuits would be evoked when such change occurs. To address this question, we performed multi-scale synapse analyses, in which dendrite and somatic events are simultaneously assessed during precise stimulation of identified spines by two-photon glutamate uncaging. Interestingly, mice with knockdown of SETD1A and DISC1, both well-established animal models for schizophrenia, exhibited a significant number of extra-large (XL) spines (corresponding to more than three standard deviations from the average of normal mice). We found XL spines evoke a marked supralinear synaptic amplification, and clustered inputs to a few XL spines were sufficient to drive neuronal firing. We experimentally and theoretically observed that the cluster density of XL spines negatively correlated with working memory, which can contribute to psychiatric pathophysiology. Furthermore, the cluster density of XL spines was significantly overrepresented in the postmortem brain of schizophrenia than age- and gender-matched control. The currently dominant hypothesis of schizophrenia pathophysiology is a reduction in spine density (the over-pruning hypothesis). However, our results presented here suggest that the hypothesis may need to be revised and that more in-depth investigations into the interactions between intra-spine and dendritic computations and their effect on brain (dys)function are needed.

Background: The pathophysiology of hikikomori has not been clarified, and biological traits that objectively characterize it remain unexplored.

Methods: Drug-free patients with hikikomori conditions (n=42) and healthy controls (n=41) were recruited. Psychological assessments for the severity of hikikomori (HQ-25) and depression (PHQ-9 and HAMD-17) were conducted. Blood biochemical tests and plasma metabolome analysis were performed. Based on the integrated information, machine-learning models were created to discriminate cases of hikikomori from healthy controls, predict hikikomori severity, stratify the cases, and identify metabolic signatures that contribute to each model.

Results: Long-chain acylcarnitine levels were remarkably higher in patients with hikikomori; bilirubin, arginine, ornithine, and serum arginase were significantly different in male patients with hikikomori. The discriminative random forest model was highly performant, exhibiting an area under the ROC curve of 0.854. To predict hikikomori severity, a partial least squares PLS-regression model was successfully created with high linearity and practical accuracy. Additionally, blood serum uric acid and plasma cholesterol esters contributed to the stratification of cases.

Conclusions: These findings reveal the blood metabolic signatures of hikikomori, which are key to elucidating the pathophysiology of hikikomori. In addition, we believe that these data will shed new light on considering the biological meanings of social-distance in the COVID-19 era.

We recently defined that CaMKIV null mice exhibit drug-resistant depression behaviors. In CaMKIV null mouse reveals increased hippocampal adult neurogenesis which is correlated with depression. We examined whether stimulation of sigma-1 receptor (Sig-1R) improves depressive-like behaviors and hippocampal adult neurogenesis in both wild-type and CaMKIV null mice. Sig-1R is molecular chaperone regulating calcium efflux from the neuronal endoplasmic reticulum to mitochondria. Repeat treatment of dehydroepiandrosterone (DHEA), endogeneous Sig-1R legand, significantly ameliorated decreased hippocampal adult neurogenesis and depressive-like behaviors in olfactory bulbectomized mice. Next, we focus on depressive-like behaviors in CaMKIV null mice. Repeated stimulation of Sig-1R by treatment with the agonist SA4503 or the SSRI fluvoxamine for 14 days improves depressive-like behaviors and hippocampal adult neurogenesis in CaMKIV null mice. By contrast, treatment with paroxetine, which lacks affinity for Sig-1R, did not alter these behaviors and hippocampal adult neurogenesis. Taken together, Sig-1R stimulation increased hippocampal adult neurogenesis, which is closely associated with therapeutic target of drug-resistant depression.

Microglia, the resident immune cells of the brain, play essential roles in neuronal development, homeostatic function, and neurodegenerative disease. Human microglia are very different from mouse ones, and thus research of mouse microglia may not always represent the response of human microglia. Further, most research on human microglia is performed in vitro, which does not accurately represent microglia characteristics under in vivo conditions. To address these issues, we developed a simplified method to generate induced pluripotent stem cell-derived human microglia (iPSMG) and transplant them non-invasively into the brain via a transnasal route in immunocompetent mice. We show that by combining pharmacological ON/OFF of a colony stimulating factor 1 receptor antagonist PLX5622 and intranasal transplantation, iPSMG can be non-invasively transplanted into the mouse brain; the transplanted microglia can migrate to each brain site, proliferate and be engrafted for at least 60 d without any immunosuppressants. The relationship between transplanted iPSMG and endogenous mouse MG varies depending on the brain region and transplanted iPSMG mature over a month within the mouse brain. Our method provides a way to non-invasively transplant cells into mouse brain and may therefore be valuable for evaluating how human microglia affect physiological and pathological brain functions.

Microglial activation is crucial for stress pathology in mental illness. However, how stress alters microglia remains elusive. Here we found that microglia show brain region-specific (local) and non-specific (global) transcriptional responses to chronic social stress in mice. Local super-enhancer responses specific to the medial prefrontal cortex (mPFC) emerged after acute stress leading to adjacent gene expressions after chronic stress. Global super-enhancer responses that occurred in both the mPFC and nucleus accumbens emerged along with adjacent gene expressions after acute and chronic stress. We are investigating transcription factors involved in the local and global responses. These findings pave the way for elucidating a transcriptional program of microglia for integrating brain and peripheral signals under stress. In this symposium, I will introduce these recent findings and discuss their implications in the glial role for brain-peripheral interactions.

Cancer cachexia affects many patients with terminal cancer and significantly reduces their quality of life. Enhanced neuroinflammation in the hypothalamic region, which plays an important role in the peripheral-central linkage, has recently been reported to contribute to the development of various diseases. In addition, the activity of non-autonomous cells, such as glial cells, may be responsible for the variability of nerve activity seen in systemic diseases. In this study, we investigated the functional changes in glial cells of the hypothalamus under stress, inflammation and cancer cachexia using magnetic-activated cell sorting. We found changes in the mRNA expression of inflammatory cytokines and immune checkpoint-related molecules in microglia isolated from stress-challenged and cachexia model mice as well as LPS-induced inflammation model, whereas the mRNA levels of stress response-related factors in astrocytes were altered in these models. These results suggest that systemic inflammation and cancer cachexia may induce functional changes in hypothalamic glial cells similar to excess stress pathology and such synchronized phenomenon may at least partly correspond to the exacerbation of cancer-related negative conditions.

Histamine plays an important role in the control of brain functions. Our recent study showed that histamine N-methyltransferase (HNMT), a histamine-metabolizing enzyme, regulates brain histamine concentration. Although previous study indicated the expression of HNMT in neurons, the contribution of neuronal HNMT to brain histamine system is still unknown. In the present study, we phenotyped neuron-specific Hnmt knockout (cKO) mice to clarify the importance of neuronal Hnmt. First, we generated cKO mice by injecting AAV9-hSyn1-Cre, which express Cre recombinase specifically in neurons, to Hnmt flox mice. Histamine concentration of cKO brains was significantly elevated compared to that of control brains, indicating the importance of neuronal Hnmt for brain histamine concentration. Behavioral test battery demonstrated that cKO mice showed reduced anxiety-like behaviors, reduced depression-like behaviors and increased locomotor activity in novel and familiar environment. These data show the importance of neuronal Hnmt for brain histamine system and brain functions.

A whole-cell patch-clamp study was carried out to elucidate serotonin (5-HT)-induced modulation of excitatory synaptic transmission onto cholinergic neurons in the basal forebrain (BF) using brain slices obtained from young rats (P12-20). BF cholinergic neurons were identified with Cy3-192IgG. Excitatory postsynaptic currents (EPSCs) were evoked by focal stimulation. 5-HT, 8-OH-DPAT (DPAT), a 5-HT_1A receptor agonist or CP93129 (CP), a 5-HT_1B receptor agonist inhibited the amplitude of EPSCs. 5-HT-induced inhibition was mostly antagonized in the presence of both 5-HT_1A and 5-HT_1B receptor antagonists. Paired-pulse ratio (PPR) and coefficient of variation (CV) of the EPSCs were increased by CP, whereas DPAT had no effect on PPR or CV. DPAT inhibited the inward currents induced by puff application of L-glutamate, whereas CP had no effect. 5-HT-induced inhibition was decreased in the presence of ω-agatoxin TK (Aga) compared to that without Aga. Furthermore, CP-induced inhibition of EPSCs was eliminated in the presence of Aga, whereas DPAT still inhibited the EPSCs even in the presence of Aga. These results suggest that 5-HT_1A receptors reduce the sensitivity of postsynaptic glutamate receptors, whereas 5-HT_1B receptors presynaptically inhibit glutamate release by selectively blocking P/Q-type calcium channels, thereby both inhibiting excitatory transmission onto BF cholinergic neurons.

Long-term depression (LTD) at parallel fiber to Purkinje cell (PC) synapse in the cerebellum is the cellular basis for cerebellar motor learning. Although some signaling molecules including protein kinase G (PKG) and MAP kinase (MAPK) are essential for the LTD induction, the molecular mechanism for long-term memory has not been fully understood.

8-nitro-cGMP is produced by guanylyl cyclase in the presence of nitric oxide and reactive oxygen species (ROS). In contrast to cGMP, 8-nitro-cGMP has resistance to PDE-dependent catalysis and can activate PKG for a long time. Therefore, we hypothesized that 8-nitro-cGMP-mediated signal is essential for cerebellar LTD. Application of 8-nitro-cGMPS, an analog for 8-nitro-cGMP, to PCs significantly inhibited LTD induction in acute mouse cerebellar slices. Pharmacological inhibition of ROS also abolished LTD induction, suggesting involvement of ROS/8-ntiro-cGMP signals in cerebellar LTD.

8-nitro-cGMP activates protein kinase G (PKG), and our previous studies indicate PKG activate extracellular signal related kinase (ERK), a type of MAPK. We therefore examined involvement of ERK in cerebellar LTD, using mutant mice lacking ERK 1 and/or 2. The LTD was impaired in the ERK 1&2 double-knockout cerebellum. There results indicate essential role of 8-nitro-cGMP – ERK signals in cerebellar LTD.

Accumulating evidence suggests that molecular dynamics at nanometer scale is crucial for brain functions and disorders. Single-molecule fluorescence imaging is a super-resolution live imaging method that enables direct tracking of movement of individual molecules. However, conventional single-molecule imaging has been applicable only to dissociated cells on coverslips due to technical limitations, preventing the analysis of events that occur only in the intact brain tissue. In this study, we set out to develop a method for single-molecule imaging within brain slices and the brain in vivo. We developed and employed a novel chemical tag technology named De-QODE. This technology consists of a small-molecular QODE probe and DeQODE protein tag. Non-fluorescent QODE becomes highly fluorescent upon reversible binding to DeQODE. These properties allow us to lower background and avoid photobleaching even in light-scattering tissue samples. We succeeded in continuous and high-density tracking of QODE molecules activated by membrane-tagged DeQODE in pyramidal neurons deep within acute cortical slices. This result indicates that our De-QODE-based method is highly promising to realize the pharmacology based on single-molecule dynamics.

Oxytocin (OT), a hypothalamic neuropeptide that acts as a neuromodulator in the brain, orchestrates a variety of animal behaviors. However, the relationship between brain OT dynamics and complex animal behaviors remains largely elusive, partly because of the lack of a suitable technique for its real-time recording in vivo. Here, we describe a G protein-coupled receptor-based green fluorescent OT sensor with a large dynamic range, optimal affinity, ligand specificity to OT orthologs, minimal effects on downstream signaling, and long-term fluorescence stability. By combining viral gene delivery and fiber photometry-mediated fluorescence measurements, we demonstrated the utility of the sensor for real-time detection of brain OT dynamics in living mice. Importantly, our measurements revealed "OT oscillation," a hitherto unknown rhythmic change in OT levels in the brain. The new fluorescent OT sensor will allow the analysis of OT dynamics in a wide variety of physiological and pathological processes.

Liver dysfunction including liver cirrhosis and hepatocellular carcinoma (HCC) often causes osmolyte and water imbalance such as edema and ascites. However, the feature and mechanism of liver injury-mediated body fluid dysregulation remain to be elucidated. In the present study, we examined the effect of HCC on body sodium and water balance in rats. Male Wistar rats were administered diethylnitrosamine, a carcinogenic drug, for 8 weeks to establish HCC. Three weeks after the cessation of diethylnitrosamine administration, we evaluated body mass, sodium, and water balance. Compared with control rats, HCC rats reduced body mass and the amount of total body sodium and water. HCC rats also showed enhanced glucocorticoid receptor activity in skeletal muscle, a marker of catabolism. On the other hand, relative tissue sodium and water content per skin and muscle tissue weight were significantly increased in HCC rats. These HCC-induced changes in sodium and water balance were significantly associated with increased 24 hours urinary aldosterone excretion and increased urea osmolyte-driven renal water conservation. These findings suggest that HCC induces osmolyte and water retention at the tissue level in parallel with body mass loss and that enhanced glucocorticoids, aldosterone, and the urea-driven renal water conservation system lead to these HCC-induced changes. The tissue sodium and water retention accompanied by body mass loss may be a causative factor for osmolyte and water imbalance in liver failure.

Kidney has ability to compensate its size and function against the nephron loss for maintaining total renal function, for example, in both donor and recipient in renal transplantation. However, the factors that regulate this compensation have not been fully clarified yet. It has been reported that approximately 70% of renal transplantation recipients suffer from anemia. Hereby we examined the effects of anemia on the compensatory renal hypertrophy in the mice lacking erythropoietin production. The anemic mice showed disrupted compensation after UNX compared to normoxemic mice. The disruption was accompanied by the sustained phosphorylation of ribosomal protein S6, a marker of mTOR activation, and by the sustained activation of YAP, a key transcriptional factor for the organ development; both of which had been normalized after successful compensation in the normal mice. There were no difference in the numbers of Ki67- and TUNEL-positive cells and in the capillary blood flow between anemic and normoxemic mice. In conclusion, anemia disrupted compensatory renal hypertrophy after UNX despite the activated tissue growth signals.

Ischemic acute kidney injury (AKI) is a serious renal dysfunction caused by surgical invasion and transplantation. Proximal tubular damage is known to be the main characteristics of pathological progression in ischemic AKI. However, the involvement of distal tubule in ischemic AKI remains well unknown. We have previously shown that Na⁺/Ca²⁺exchanger type 1 (NCX1) is expressed on the basolateral side in distal convoluted tubule and is involved in urine production and electrolyte exertion. In this study, we evaluated the pathophysiological significance of distal tubular NCX1 in ischemic AKI, using distal tubular-specific NCX1 deficient (NCX1-cKO) and NCX1 transgenic (NCX1-TG) mice. We subjected these mice and wild-type mice to sham surgery or 30 min of unilateral renal ischemia and reperfusion (IR) with contralateral nephrectomy. BUN and serum creatinine were significantly increased in both NCX1-TG mice and wild-type mice after IR injury. In contrast, these increases were remarkably suppressed in NCX1-cKO mice after IR injury. The histological findings showed that the distal tubular damages after IR injury were alleviated in NCX1-cKO mice, but were exacerbated with macrophage infiltration to renal medulla in NCX1-TG mice. These results suggest that distal tubular NCX1 plays a critical role in the pathophysiology of ischemic AKI.

Sustained mechanical stresses (e.g. high blood pressure) damage the renal glomerulus resulting in proteinuria. Podocytes express the canonical transient receptor potential 6 (TRPC6) channel, the activity of which is modulated by receptor and mechanical stimulations in a complex manner. However, the implication of this modulation for the glomerular barrier function is rather poorly elucidated. To address this point, we carried out the albumin-permeation assay using immortalized mouse podocytes stably expressing the wild-type (wt) TRPC6 or its gain-of-function mutant associated with focal segmental glomerulosclerosis (FSGS), M131T. Differentiated podocytes were grown on cell-culture insert membranes (pore size: 0.4micrometer). Compared to unstimulated or angiotensin II (Ang II) alone, simultaneous stimulation with Ang II and a membrane-expanding agent 2,4,6-trinitrophenol (TNP) reduced the leak of FITC-labelled albumin across the membranes in both wt-TRPC6- and M131T-expressing podocytes. SAR7334, a potent TRPC6-specific inhibitor, increased the albumin leak, the effect being more prominent in the latter. These results suggest that TRPC6 activation by simultaneous receptor and mechanical stimulations may reinforce the filtration barrier function mediated by podocytes, and this may be impaired by the FSGS-associated mutation M131T.

[Background]

IL-6 family cytokines play protective roles in cardiomyocytes via STAT3 activation. Glomerular podocytes, as an important component of the kidney blood filtration, are terminally differentiated cells as well as cardiomyocytes. Although some studies have shown that STAT3 activation is associated with podocyte dysfunction, it remains unclear whether activated STAT3 exhibits differential functions depending on cytokines. The aim of this study is to assess the effects of IL-6 family cytokines/STAT3 signaling in podocytes.

[Methods and results]

To examine the pathophysiological relevance of IL-6 family cytokines in kidney diseases, C57BL/6J mice were subjected to ischemia-reperfusion or lipopolysaccharide (LPS) treatment. Quantitative PCR demonstrated that the expression level of IL-6, leukemia inhibitory factor (LIF) and IL-11 was upregulated in injured kidneys. In cultured podocytes, STAT3 was rapidly activated in response to the stimulation with IL-6, LIF or IL-11. Interestingly, LIF and IL-11 treatment suppressed H₂O₂-induced cell death in cultured podocytes, whereas IL-6 tended to increase cell death.

[Conclusion]

STAT3 could differentially function in an activator cytokine-specific manner, in podocytes, providing the important information for the development of therapy targeting STAT3 for kidney diseases.

Ketamine, an N-methyl-D-aspartate (NMDA) receptor antagonist, is the prototype for a potential new generation of glutamate-based antidepressants that rapidly relieve symptoms of depression within hours of treatment. Studies in rodents have demonstrated that neuroplasticity in the medial prefrontal cortex (mPFC) is critical for the antidepressant actions of ketamine. However, effector circuits downstream of the mPFC underlying the rapid antidepressant responses remain unknown. To address this issue, we used optogenetic and chemogenetic circuit mapping in rodent models for studying depression, demonstrating the role of the basolateral amygdala (BLA) and bed nucleus of stria terminalis (BNST) as downstream targets of the mPFC mediating distinct behavioral effects of ketamine. By inhibiting isolated mPFC projections in the period immediately following ketamine administration, we found that mPFC-mediated activation of BLA principal neurons, and subsequent projections to the ventral hippocampus, mediate a subset of ketamine's persistent antidepressant-like effects on passive coping behavior but not on anxiety-like and reward-seeking behaviors. In contrast, mPFC projections to the BNST are necessary and sufficient to produce persistent antidepressant-like effects on anxiety-like and reward-seeking behaviors but not on passive coping behavior. Therefore, our data support a model where distinct downstream circuits of the mPFC contribute to producing separate antidepressant-like behavioral responses.

Sleep is closely related with mental health, and affected by drug therapy in psychiatric disorders, and vice versa. The delta opioid receptor (DOR) agonist KNT-127 has been reported to have anxiolytic effects. Unlike benzodiazepines, KNT-127 has no side effects such as dizziness and amnesia. However, its effects on sleep have not been studied. In the present study, we investigated the effects of KNT-127 on sleep in the light period in mice. The vigilance states (e.g., wakefulness, rapid eye movement (REM) and non-REM sleep) of the ddY-mice (6-10 weeks) were classified based on the hippocampal local field potential (LFP) and neck muscle electromyogram. KNT-127 (3-30 mg/kg, i.p.) dose-dependently decreased the mean REM and non-REM sleep period, and prolonged the mean wakefulness period during 5 hr after its injection. At the wakefulness and REM sleep periods after the KNT-127 treatment, the gamma power and theta peak frequency were increased in the hippocampal LFP, suggesting that KNT-127 enhanced the neuronal activities. Pre-treatment of naltrindole (10 mg/kg, s.c.), a DOR antagonist, prevented the KNT-127-induced decrease in non-REM, but not REM, sleep. Naltrindole alone did not influence the vigilance states. Together, KNT-127 increased wakefulness by decreasing non-REM and REM sleep via DOR-dependent and -independent mechanisms, respectively.

Kynurenine 3-monooxygenase (KMO) is a kind of rate-limiting enzyme in the kynurenine pathway. We investigated the change in KMO expression and intermediary metabolite levels after intracerebral hemorrhage (ICH) in neuronal injury. Treatment with thrombin to primary-cultured microglia increased the KMO expression through the p38 MAPK pathway. In the cultured medium, the ratio of quinolinic acid (QUIN), an N-methyl-D-aspartate receptor agonist, to kynurenic acid (KYNA), its antagonist, was increased, whereas the level of 3-hydroxykynurenine, a redox-active compound, showed no significant change. The increased QUIN/KYNA ratio was blocked by Ro61-8048, a KMO inhibitor. In the mouse ICH model, immunohistochemical staining showed that KMO was co-localized with neurons, microglia, and astrocytes. The QUIN/KYNA ratio was increased after ICH, but blocked by the intracerebroventricular injection of Ro61-8048 or liposomal clodronate, a microglia toxin. Ro61-8048 ameliorated the loss of neurons, as indicated by NeuN-immunopositive cells, at the perihematomal region and repaired their abnormal behaviors without affecting the hematoma size. In conclusion, thrombin-induced alterations of microglial KMO and intermediary metabolites of the kynurenine pathway were suggested to play important roles in neuronal injury after ICH.

It is recently known that early brain injury is one of the important pathophysiology to determinant the prognosis of subarachnoid hemorrhage (SAH). We herein examined the role of sphingosine related pathway activation in the treatment of early brain injury of experimental SAH model.

SAH were induced by endovascular perforation in mice or rats, and they were treated with 1) unfractionated heparin 2) isoflurane 3) FTY720 (sphingosine receptor agonist). The animals evaluated neurological scores, brain edema and sphingosine metabolism-related molecular markers.

The above-mentioned treatments improved neurofunction and brain edema, and provided antiapoptotic effects such as upregulation of phosphorylated Akt and downregulation of caspase-3. The effects were associated with activation of sphingosine kinase and sphingosine receptor.

We suggest that activation of the sphingosine related pathway should have beneficial effects in early brain injury of experimental SAH and could improve the prognosis of the clinical SAH patients.