SIRT1, an NAD+-dependent deacetylase, positively regulates autophagy, which maintains muscle function and declines with age. We recently reported that administration of resveratrol (RSV), an activator of SIRT1, to a mouse model of Duchenne muscular dystrophy restores muscle autophagy, preserves muscle mass, and improves exercise tolerance. Here, we hypothesized that RSV attenuates age-related loss of muscle mass and preserves muscle function by activating autophagy.

We compared exercise tolerance of aged DDY mice that had either a control diet or a diet containing RSV for 37 weeks. Although treadmill running distance and grip strength of the mouse were not changed, rotarod riding time was significantly extended in RSV-treated mouse. Myofiber diameter of tibialis anterior muscle was higher in RSV-treated mice. In the muscle of RSV-treated mice, acetylated lysine levels were reduced, suggesting that SIRT1 is activated by administration of RSV. In addition, RSV increased LC3-II protein level, a marker of autophagosomes, and decreased levels of p62 and ubiquitinated proteins, which are degraded by autophagy. These results suggest that RSV activates autophagy in the muscle.

In conclusion, we propose that RSV attenuates aging-related loss of muscle mass and motor dysfunction via activating autophagy in mice.

Objective: Skeletal muscle fiber conversion bears a part in multiple myopathies, and one of those is Sarcopenia which is known to correlate with aging, nutritional deficiency, and so on. In this study, we focused on muscle specific enriched microRNAs (myomirs) to understand the mechanisms of muscle fiber conversion.

Method & Results: In-silico study, we selected the microRNA-133a-3p (miR-133a), a myomir relevant to the skeletal myogenesis. Using the human iPS cell-derived skeletal myogenesis system, we identified that expression level of miR-133a was elevated with a peak at day 5 after the induction of myogenesis. Overexpression of miR-133a mimic or inhibitor at the beginning of myogenesis increased mRNA expression of Myh7, a specific marker of slow oxidative myotube, or Myh1, a marker of fast-type myotube, respectively. Furthermore, we found that, under the serum-deprived culture condition, overexpression of miR-133a mimic preserved the cell feature of oxidative fibers, although miR-133a inhibition deformed the tubular formation of oxidative myotubes.

Conclusion: MicroRNA-133a functioned to buttress the gene expression of oxidative fiber-specified myosin heavy chain, and to preserve myotubes from nutritional stress, suggesting the possibility that miR-133a would be a therapeutic target for sarcopenia.

[Background]

Gene transfer into the target organ is a critical process of gene therapy. Although gene transfer by physicochemical method has relatively safe, the transfer efficiency is low depending on the target organ. To develop the effective and efficient gene transfer method, we focused on Pyro-drive Jet Injector (PJI) which was developed based on pyrotechnics. The aim is to explore the effectiveness of PJI in gene transfer into muscle tissues.

[Methods/Results]

pcDNA3.1-venus-plasmid was injected to the thigh muscles of C57BL/6J mice with PJI or needle. Immunofluorescence demonstrated the venus was expressed in a wider range in PJI group than needle group. To examine the validity of PJI for gene therapy, pcDNA3.1-humanFGF2 plasmid, an angiogenic factor, was injected into the ischemic thigh muscles with PJI or needle. ELISA showed the FGF2 protein was increased in the thigh muscle tissue in PJI group. Significantly, histological analyses revealed the muscle cell cross-sectional area and endothelial marker CD31 (+) cells was increased in the PJI-FGF2 group compared with needle-FGF2 or PJI-control plasmid. Finally, PJI method was successful in gene transfer into murine heart with high efficiency when compared to needle method.

 [Conclusion]

Gene transfer by PJI may be a useful approach for the treatment of muscle diseases.

Mastic is a natural resin collected from the Mastic tree, which exists only on the Greek island of Chios. Our previous study has shown that oral care with mastic gel in dogs significantly reduces the activity of periodontal disease bacteria, however, scientific evidence of mastic efficacy still has to be examined. The aim of this study is to identify the novel efficacy of Mastic by focusing on the anti-allergic effect in a mouse model of atopic dermatitis (AD). A mouse mode of AD was generated in female NC/nga mice by repetitive topical sensitization with toluene diisocyanate (TDI). Topical treatment of 3% or 5% Mastic gel daily onto ear auricle and back skin during the experiment. Thickness of back skin, AD score, trans epidermal water loss (TEWL), and itch behavior were measured weekly. One day after the last TDI exposure, all mice were euthanized and serum, auricular lymph node and skin tissue were sampled for histology, cytokine determination and FACS analysis. Mastic treatment significantly attenuated thickness of skin, AD score, TEWL and itch behavior. Corroborated reduction was seen in the numbers of T cells and IgE-B cells as well as pro-inflammatory cytokine production. Our findings demonstrated that topical Mastic treatment significantly ameliorates the inflammatory and pruritic responses in a mouse model of AD.

Accumulating evidence has shown that release of inflammatory cytokine Interleukin (IL)-1α/β is regulated by Nod-like receptor-dependent (canonical) and -independent (non-canonical) inflammasome. Caspase-11, the key component of non-canonical inflammasome, senses bacterial lipopolysaccharides (LPS) in the cytosol to induce proteolytic cleavage of IL-1α/β and Gasdermin D. Subsequently, processed mature IL-1a/b is released through the pore on the plasma membrane that is generated by fragmented active Gasdermin D. Although excessive non-canonical inflammasome activation often causes various inflammatory diseases such as endotoxin shock, non-canonical inflammasome inhibitor has not been well identified yet. In this study, we performed screening of 201 bioactive lipids to identify 15d-prostaglandin J2 (PGJ2) as the novel non-canonical inflammasome inhibitor. Mass spectrometry analysis of 15d-PGJ2 binding proteins revealed the target of 15d-PGJ2 that is involved in caspase-11 activation. Furthermore, we found that sequential regulation of 15d-PGJ2 production by Toll-like receptor 4 and non-canonical inflammasome upon LPS stimulation. Finally, we provided in vivo evidence that PGJ2 treatment ameliorates septic shock in mice. These findings suggested that 15d-PGJ2 is the negative feedback inhibitor of the non-canonical inflammasome. Therefore, 15d-PGJ2 could be an effective medication for the treatment of inflammatory diseases such as endotoxin shock.

For a long time ago, people have believed that good medicine tastes bitter to the mouth; however, whether the bitter taste itself has therapeutic effects is less studied. Generally, bitter taste is recognized by Type-2 bitter-taste receptors (TAS2Rs) belonging to G-protein coupled receptors and TAS2Rs are localized on taste bud cells of the tongue. Growing evidence suggests that TAS2Rs are expressed not only in the taste bud cells but also in other cells including airway smooth muscle cells, intestinal tuft cells and immune cells. In this study, we show that bitter taste substance-TAS2R axis regulates neutrophil migration. By gene expression analysis, we found that neutrophils express TAS2R126, TAS2R135 and TAS2R143. Next, we observed the effect of TAS2R126/135/143 agonists on neutrophil migration. Although TAS2R135 agonists did not affect neutrophil migration, TAS2R126/143 agonists significantly enhanced CXCL2-induced neutrophil migration. The enhancing effects were not observed in a TAS2R126/143 deficient neutrophil-like cell line. In addition, TAS2R126/143 agonist also promotes neutrophil infiltration into zymosan-injected abdominal cavity. These results suggest that TAS2R126/143 signaling facilitates neutrophil-mediated immune responses and may be targets to promote host defense against infection.

Aquaporins are the water channel that facilitate water transport across plasma membrane. In human, there are 13 AQPs, at least one of which is found in every organ system. Recently, it is getting clear that AQPs regulate not only water metabolisms, but also various cellular functions, such as growth and migration. We have found that AQP5, a major isoform in lung epithelia, enhances TNF-α-induced-chemokine expression, but suppresses the response to Th2 cytokines in vitro. It has also known that the expression of AQP5 is markedly reduced in mice with asthma, suggesting that the change of expression level of AQP5 may contribute to the pathogenesis of inflammatory respiratory disease. In this study, therefore, we have established a transgenic (Tg) mouse in which AQP5 is highly expressed specifically in the lung epithelial cells, and examined Th2 inflammatory responses in a model of asthma. In the Tg-mice, the inflammation, assessed by cells in BALF and chemoattractant expression, was considerably less than that in wild type mice, whereas mucus production was increased in Tg-mice. Additionally, mRNA expression of Th2 cytokines were decreased in Tg-mice. Our results indicate that AQP5 can suppress allergic inflammation in vivo, and suggest that decreased expression of AQP5 may suppress the development of inflammatory pathology.

Invariant natural killer T (iNKT) cells produce large amounts of cytokines to induce an inflammatory or reparative function. In addition, NKT cells are highly enriched in mice liver. The objective of the present study was to examine whether iNKT cells contribute to liver repair following hepatic ischemia and reperfusion (I/R) in mice. Wild-type (WT) mice and CD1d-deficient (CD1d^-/-) mice were subjected to hepatic I/R. Compared with WT mice, CD1d^-/-mice showed delayed liver repair as indicated by increases in ALT levels and hepatic necrotic area and by decreases in PCNA expression. These were associated with decreases in mRNA levels of IL-4 and IL-13 and in accumulation of Ly6C^low reparative macrophages in the liver. By contrast, treatment of WT mice with α-galactosylceramide (α-GalCer) at 0 h post-reperfusion stimulated liver repair after hepatic I/R. At 72 h post-reperfusion, iNKT cells and Ly6C^low macrophages were accumulated in the liver treated with α-GalCer, which were associated with enhanced expression of IL-4 and IL-13. These results suggest that iNKT cells and reparative macrophages are involved in promotion of liver repair after hepatic I/R injury.

Oral administration of a food-derived hydrophilic amino acid ergothioneine (ERGO) enhances memory function in mice at clinically achievable plasma concentration, although the mechanisms underlying the ERGO-induced cognitive enhancement remain unclear. We here tried to clarify the mechanisms using mice fed ERGO-free diet (ERGO-deficient mice). Hippocampal ERGO concentration in the ERGO-deficient mice was about 15 times lower than that in mice fed with normal diet. Oral administration of ERGO in ERGO-deficient mice enhanced learning and memory according to novel object and spatial recognition tests with a concomitant restoration of ERGO level in the hippocampus. To clarify the mechanisms, we focused on hippocampal neurogenesis induced by tropomyosin receptor kinases B (TrkB), one of the neurotrophin receptors. The oral ERGO administration increased area of a neurogenesis marker doublecortin-positive cells in the hippocampal dentate gyrus with a concomitant increase in protein expression of phosphorylated TrkB. In contrast, simultaneous administration of a TrkB inhibitor significantly suppressed the ERGO-induced cognitive enhancement and neurogenesis. These results suggest that the ERGO-induced cognitive enhancement would be caused by phosphorylation of TrkB and promotion of neurogenesis in the hippocampal dentate gyrus.

Ninjinyoeito (NYT), a traditional Japanese medicine, is effective on physical strength, fatigue and anorexia. Recently, NYT ameliorated a cognitive dysfunction in patients with Alzheimer's disease (AD), however the mechanisms have been still unclear. In this study, we investigated the effects of NYT on spatial memory impairment in dementia model of rats. The dementia model of rats were prepared with transient cerebral ischemia and intraventricular injection of β-amyloid 1-42 for 7 days (CI+Aβ). NYT or donepezil were orally administered for 7 days after the cerebral ischemia. Spatial memory was evaluated using Morris water maze. We investigated the hippocampal and prefrontal cortical protein expression of α-amino-3-hydroxy-5-4-isoxazole propionic acid (AMPA) receptor subunit GluA1 and GluA2, Ca²⁺/calmodulin-dependant protein kinase Ⅱ (CaMKⅡ), and phosphor-CaMKⅡ. The treatment with NYT in the CI+Aβ rats shortened the extended time to reach the platform in Morris water maze, as well as donepezil treatment. Although NYT treatment did not ameliorate the decrease in GluA1 and GluA2 expression levels in hippocampus, NYT treatment showed the increase in GluA1 expression level and phoshor-CaMKⅡ/CaMKⅡ ratio in the prefrontal cortex of CI+Aβ rats. Therefore, NYT might improved spatial memory impairment via facilitation of glutamate signaling in the prefrontal cortex of the CI+Aβ rats. Our results suggest that NYT is a useful treatment for AD patients.

Reelin protein, an extracellular matrix protein, plays an important role in embryonic neuronal migration and layer formation in cerebral cortex. In the adult brain, reelin is produced by GABA-ergic interneurons and affect synaptic plasticity, dendritic morphology and cognitive function. We have recently found a schizophrenia patient carrying a novel exonic deletion in RELN gene by CNVanalysis on a mainly Japanese population. Meanwhile touchscreen-based operant tasks have been developed for rodents to provide a better translational approach across species for further understanding of the cognition and attentionimpairment observed in various neuropsychiatric disorders. In the present study, we aimed to explore the performance of RELN deletion mice (Reln-del), using touchscreen-based visual discrimination (VD) and 5-choice serial reaction time (5-CSRT) tasks. Mice were initially trained to discriminate stimuli simultaneously displayed on the screen and received a liquid reward. The cognitive impairment in Reln-del mice was demonstrated by an increase in the number of total trials,total errors trials, total correction trials and total sessions in complex VD taskand in complex reversal learning. On the other hand, they showed no significant impairments in 5-CSRT task. These results suggest that the exonic Reln deletion lead to an impairment in cognitive function but not in attentional processes.

We have recently found that nicotine (Nic) enhances object recognition memory (ORM) in the novel object recognition test (NOR) via stimulation of α4β2 and α7 nicotinic acetylcholine receptors (nAChRs) in the medial prefrontal cortex (mPFC) of mice. Additionally, in vitro electrophysiological analyses revealed that Nic increases firing activity of mPFC layer V pyramidal neurons, which was occluded by 4-aminopyridine (4-AP), a non-selective voltage-dependent potassium (Kv) channel inhibitor, or XE-991, a selective Kv7 channel inhibitor. According to these findings, we hypothesized that Nic increases mPFC neuronal activity by suppressing Kv channels, resulting in the enhancement of ORM. To test this hypothesis, we first observed that c-Fos expression is increased in the mPFC after systemic Nic administration and that suppression of mPFC neuronal activity with inhibitory DREADD significantly inhibits the Nic-induced ORM enhancement, indicating the importance of mPFC neuronal activity in ORM enhancement. Moreover, we found that intra-mPFC injection of 4-AP or XE-991, as well as Nic, enhances ORM. Since one of the targets of 4-AP is Kv4.3 channels, we tested the effects of NS5806, a Kv4.3 channel activator, on Nic-induced ORM enhancement and found that intra-mPFC injection of NS5806 suppressed the Nic-induced ORM enhancement. Similarly, a Kv7 channel activator retigabine also attenuated Nic-induced ORM enhancement. These data suggest that Nic enhances ORM via activation of mPFC neurons through the suppression of Kv4.3 and Kv7 channels.

Introduction

Spinal cord ischemia and reperfusion (I/R) injury is one of the most devastating complications after thoracic and thoracoabdominal aortic aneurysm surgery. Several studies showed that levels of HMGB1 in serum were increased in patients who suffered from spinal cord I/R injury. These data strongly suggest that HMGB1 may play a crucial role in spinal cord I/R injury.

Materials and Methods

Male New Zealand white rabbits were used for the experiments. The abdominal aorta at the level of the left renal artery was exposed, and, after heparinization, occluded for 11 minutes. The rabbits were intravenously administered an anti-HMGB1 mAb (#10-22, 2mg/kg) or class-matched control IgG (anti-keyhole limpet hemocyanin mAb) twice immediately and 6h after reperfusion. The neurological findings were assessed at 6, 24 and 48 hours after reperfusion. At 48hr after reperfusion, the rabbits were sacrificed to obtain the specimen of spinal cord and blood samples.

Results

Administration of anti-HMGB1 mAb ameliorated the intensity of spinal cord infarction and preserved the number of motor neuron cells, in association with decreased activated microglia and astrocyte. Consequently, anti-HMGB1 mAb significantly improved the neurological outcomes after spinal I/R injury in rabbits.  These results strongly indicate that HMGB1 plays a critical role in the development of spinal cord I/R induced secondary injury through the amplification of inflammatory response.

Conclusion

Intravenous injection of neutralizing anti-HMGB1 mAb has potential as a novel therapeutic strategy for spinal cord I/R injury.

In the ischemic stroke pathologies, there is a transition from the inflammatory phase to the phase for neural repair. So far, lipid mediators have been known to regulate inflammation after ischemic stroke; however, there is still no comprehensive study which investigates various fatty acids and their metabolites in ischemic stroke. The aim of this study is to identify novel lipid which promotes neural repair after ischemic stroke.

The LC/MS analysis of the lipids collected from murine ischemic brains revealed that the polyunsaturated fatty acids and their metabolites in the brain were dramatically altered over time after the onset of ischemic stroke. We then investigated the time-dependent expression of various PLA2 subtypes which liberate free fatty acids in the ischemic brain and identified the PLA2 subtype which was important for the generation of ω-6 fatty acids, such as linoleate and gamma-linolenate. The deficiency of this PLA2 subtype exacerbated the ischemic stroke pathologies and significantly decreased the expression of peptidyl arginine deiminase which promote the neural repair after ischemic stroke. By examining various metabolites of ω-6 fatty acids, we successfully identified some lipid metabolites which induced the expression of peptidyl arginine deiminase and improved ischemic stroke pathologies.

These results indicated that a certain kind of ω-6 fatty acids was important for neural repair after ischemic stroke. The recovery from neurological deficits of ischemic stroke may be possible by the dietary intake of these neuro-reparative lipids.

Chronic cerebral hypoperfusion (CCH), resulting in an inadequate supply of blood to the brain, is manifested in various CNS diseases including neurodegenerative and mental disorders that are accompanied by cognitive impairment and associated with oxidative stress. Transient receptor potential ankyrin 1 (TRPA1), an oxidative stress-sensitive Ca²⁺-permeable non-selective cation channel, is recently found to be expressed in brain cells, although the role of TRPA1 in the CNS diseases is controversial. In this study, we investigated the pathophysiological role of TRPA1 in CCH using a mouse bilateral common carotid artery stenosis (BCAS) with 0.18 mm-diameter microcoils. At 28 days after operation, BCAS-operated mice showed cognitive impairment and white matter injury in wild-type (WT) and TRPA1-knockout (TRPA1-KO) mice. On the other hand, at 14 days after operation, BCAS-operated TRPA1-KO mice showed cognitive impairment and white matter injury, whereas these dysfunctions were not observed in WT mice. In addition, daily intraperitoneal administration of cinnamaldehyde, a TRPA1 agonist, ameliorated BCAS-induced cognitive impairment and white matter injury at 28 days after operation. These results suggest that TRPA1 plays a protective role in the development of CCH-induced cognitive impairment and white matter injury, and that TRPA1 may be a therapeutic target for CCH-related CNS diseases.

Rapid intraperitoneal water injection induces acute hyponatremia that creates an osmotic gradient driving for water entry into the brain, leading to subsequent cytotoxic edema. Aquaporin-4 (AQP4) is a most abundant water channel in central nervous system and expresses mainly in astrocyte end-feet. However, it is still controversial that how AQP4 regulates water kinetics during cerebral edema due to the lack of experimental techniques for the observation of water distribution. We established T2-weighted indirect magnetic resonance imaging (MRI) with O¹⁷-labeled water to evaluate cerebral water kinetics during edema formation in wild type and AQP4-knock out mice. We scanned coronal sections of brain along the rostral and caudal directions using 7 tesla MRI up to 40 min after the bolus injection of distilled water equal to 10% of body weight. After intraperitoneal injection of O¹⁷-labeled water, O¹⁷ concentrations in cerebral cortex, cerebellum, and lateral ventricle rapidly increased and peaked out in about 10 minutes. Peak concentrations and the time of peak concentration of O¹⁷ in WT and AQP4-KO mice were comparable respectively in each brain regions. On the other hand, the O¹⁷ elimination half-life in AQP4 KO mice was longer than that in WT mice. These results suggest that AQP4 facilitates water permeability and excretion during edema formation, which probably exacerbates the cytotoxic edema.

Brain molecular imaging with positron emission tomography (PET) has been utilized for clinical study, diagnosis and drug development of neurodegenerative diseases. However, further development of typically used ¹⁸F-labeled small compounds have been troubled by non-specific binding and low affinity against target molecules. Hence, protein binders like antibodies or their derivatives have attracted interest with their high specificity and affinity. However, their large molecular size and small entry into the brain remain challenges for clinical application. In the present study, we use a brain shuttle peptide concept for protein brain delivery and evaluate the pharmacokinetics of a blood-brain barrier-permeable small-size protein affinity ligand ([¹⁸F]AS69-ApoE). We found that [¹⁸F]AS69-ApoE showed higher brain accumulation than its original ([¹⁸F]AS69) at 10-min and 30-min post injection; on the other hand, [¹⁸F]AS69-ApoE was cleared from brain at 120-min post injection. Moreover, in vitro and in vivo stability analyses showed that these protein tracers are mostly stable in mouse plasma. These results imply the possibility of small-size protein binders for molecular brain PET imaging.

Astrocytes support the health of the brain, but reactive astrocytes are strongly associated with various neurodegenerative diseases., including the neurotoxic A1 astrocyte and neuroprotective A2 astrocyte, and an increase in their density has been demonstrated in Alzheimer’s disease brains. Positron-emission tomography (PET) is a nuclear imaging technique, making it possible to follow-up changes in astrocytic activation in vivo, thereby tracing disease progression. Monoamine oxidase B (MAO-B) is one of the markers for imaging astrocytes, whose activity is expressed in plaque-associated astrocytes. Recently, we developed  a selective and reversible tracer for MAO-B called [¹⁸F]SMBT-1. Besides, [³H]BU99008 is a tracer designed for another astrocyte marker called imidazoline 2 binding sites (I2BS). Therefore, the aim of this study was to characterize which phenotype of astrocyte dose MAO-B exist in and whether [¹⁸F]SMBT-1 can be used for imaging astrocytes.

Double immunostaining of MAO-B and C3d/S100A10, which is A1/A2 astrocyte marker, indicated that MAO-B was expressed in both A1 and A2 astrocytes. In vitro autoradiography for amyloid β, tau, and MAO-B in the same sections demonstrated the existence of MAO-B expressing astrocytes vicinity of amyloid plaque. Competitive binding assays with [¹⁸F]SMBT-1 or [³H]BU-99008 showed strong affinity of both tracers for human postmortem brain homogenates but different binding sites. Regional binding distribution  revealed a stronger correlation between [¹⁸F]SMBT-1 and GFAP than [³H]BU-99008 and GFAP.

In conclusion, MAO-B is expressed in both A1 and A2 astrocyte, and [¹⁸F]SMBT-1 is a useful PET tracer for imaging reactive astrocytes.

Single-particle tracking (SPT) is promising for precise analysis of spatiotemporal dynamics of proteins in living cells. Nonspecific fluorescent labeling and photobleaching of fluorescence in conventional SPT methods disturb reliable analysis. Here we aimed to develop a SPT technique for precise analysis of protein dynamics in living cells. We employed a regenerative fluorescence labeling technique named DeQODE technology. In this technology, a small probe molecule consisting of a fluorescent moiety and a quencher moiety is designed to reversibly bind to a protein tag named DeQODE tag which is based on a single-chain antibody against the quencher moiety. Binding of the small probe molecule to DeQODE tag turns on fluorescence. Repeated binding and dissociation of the probe to the intracellularly expressed DeQODE tag fused with the target protein in the presence of low-concentration probe molecule enables long-term SPT. To demonstrate the proof of the concept, we performed SPT of syntaxin 1A, a component of the SNARE complex, in COS7 cells, INS-1 cells, and cultured hippocampal neurons. In these cells expressing DeQODE tag-fused syntaxin 1A, we successfully obtained SPT data for hours. These results suggest that the DeQODE-based SPT is useful for precise molecular dynamics analysis in physiological and pharmacological responses.

Adenosine 5’ triphosphate (ATP) works as an extracellular signaling molecule in the brain, and is involved in regulation of neuronal activity and blood flow. However, the spatiotemporal dynamics of extracellular ATP are not fully understood due to lack of appropriate techniques, and their effects on cellular activity in brain disorders remain elusive. To explore the roles of ATP signaling, we developed a fluorescent ATP sensor, which was designed for detection of extracellular ATP. We applied the ATP sensor to the cerebral cortex of living mice, and succeeded in visualizing extracellular ATP dynamics in vivo. Using the ATP imaging technique, we visualized a wave of extracellular ATP release propagating across the cortex during pathological neuronal activity associated with brain ischemia. We compared the dynamics of the wave-like ATP release with that of vascular diameter changes during the neuronal activity, and found that the elevation of extracellular ATP concentration is accompanied by contractile response of pial arteries. These results suggest that the wave-like ATP release is involved in the regulation of vascular tone in brain ischemia. Thus, extracellular ATP imaging will contribute to elucidation of pathophysiological mechanisms and to development of therapies for brain disorders.

The initiation mechanism of drug-induced arrhythmia has been explained by EAD occurring under prolonged action potential (AP) due to I_Kr block. Thus, I_Kr block potency and AP prolongation have been used in drug screening to predict drug-induced arrhythmia. However, even if I_Kr blockers cause the prolongation in the same level, they may have different risks for drug-induced arrhythmia. For example, amiodarone prolongs AP but does not induce drug-induced arrhythmias and is considered as a relatively safe drug in clinical practice. On the other hand, terfenadine and bepridil prolong APs and induce drug-induced arrhythmia. This different risk among I_Kr blockers implies that additional mechanisms may contribute to induction and suppression of EAD. In the present study, we studied how different block kinetics of non-selective I_Kr blockers on L-type Ca²⁺ current (I_CaL) affects occurrence of EAD. By using a mathematical model of the human ventricular action potential, we found that the different drug block kinetics on I_CaL can account for the different occurrence of EAD. Voltage-independent I_CaL block suppressed EAD under prolonged AP. The I_CaL block model of amiodarone also suppressed EAD effectively. In contrast, the I_CaL block models of terfenadine and bepridil increased the occurrence of EAD. These results suggest that, to predict drug-induced arrhythmia, not only I_Kr block and AP prolongation but also voltage-dependent property of I_CaL block should be checked.

Introduction: Anti-influenza drug oseltamivir has been reported to suppress atrial fibrillation (Af) in vitro; however, since information of oseltamivir is lacking regarding in vivo anti-Af property, we studied it using the paroxysmal Af canine model.

Methods: Oseltamivir in doses of 3 and 30 mg/kg/10 min was intravenously infused to the isoflurane-anesthetized, chronic atrioventricular block dogs (n=4) under monitoring electrophysiological variables, in which Af was induced by 10 s of burst pacing on atrial septum.

Results: The high dose of oseltamivir decreased sinoatrial/idioventricular rate and mean blood pressure, whereas no significant change was observed by its low dose. Oseltamivir delayed inter-atrial conduction in dose- and frequency-dependent manners, whereas it prolonged atrial effective refractory period in dose-dependent but frequency-independent manners. The high dose prolonged ventricular effective refractory period, which was not detected by the low dose. Oseltamivir decreased Af incidence and shortened Af duration in a dose-dependent manner along with prolongation of Af cycle length.

Conclusion: Oseltamivir effectively suppressed the inducibility of paroxysmal Af and shortened its duration in vivo, which may depend on the delay in inter-atrial conduction and prolongation of atrial effective refractory period.

Introduction: Abrupt hyperglycemia induces unexpected cardiovascular responses; however, their underlying pathophysiological mechanisms remain unknown. We studied how hyperglycemia affects the cardiohemodynamic and electrophysiological variables.

Methods: Glucose in a dose of 3 g/6 mL/kg was intravenously infused over 30 min to the isoflurane-anesthetized intact dogs (n=4).

Results: Administration of glucose increased plasma glucose level and osmolality but decreased Na⁺, Cl⁻ and glucagon concentrations, whereas it tended to increase insulin level accompanied with decreased K⁺ concentration. Glucose decreased peripheral vascular resistance, but increased heart rate, left ventricular contractility and cardiac output along with increased preload to the left ventricle. Glucose infusion prolonged atrioventricular nodal and intraventricular conduction time as well as ventricular repolarization period.

Conclusion: Delay of ventricular repolarization might be partly caused by insulin-induced hypokalemia in the hyperglycemia dog model. Hyperosmolarity was reported to induce negative chronotropic, inotropic and dromotropic effects in vitro, which could be well counterbalanced by the reflex-mediated sympathetic tone in vivo, resulting in various phenotypes as shown in this study.

Cardiovascular diseases show significant sex differences, which has been attributed to the cardioprotective effects of estrogen. Recent studies unravel that the underlying mechanisms seem unlikely to be due to estrogen only. There is a great variability in size of these vessels in females throughout life, suggesting that there is little contribution of sex hormones. To understand the mechanisms, the difference in coronary blood vessels size in the fetal murine heart was analyzed. A newly-developed coronary arteriogram enabled us to visualize the morphology of the coronary vessels in the murine heart at embryonic day 17.5. Although no sex difference was found in averaged length of left ventricle, we found that the hearts of both sexes were divided into two groups according to their size. Thus, diameter of left coronary vessel (LCV) was measured for each group of heart sizes and compared between males and females. We found a sex difference in the larger group that male LCVs were thicker than female LCVs. Administration of a NO-donor, NOC7, reversed the sex difference. Furthermore, our microarray analysis identified 59 genes with sex differences. These results indicate that sex differences in functional morphology of LCVs exist at a late embryonic stage in mice.

The cell signaling molecules nitric oxide (NO) and Ca²⁺ regulate diverse biological processes through closely coordinated activities. However, signaling protein complexes that underlie the interplay between Ca²⁺ and NO pathways remain unclear in many tissues and cell types. Here we demonstrate the physical and functional interaction of the receptor-activated Ca²⁺-permeable TRPC5 channel with Ca²⁺-dependent endothelial NO synthase (eNOS) in vascular endothelial cells as well as HEK293 cells overexpressing these proteins. Upon stimulation of G-protein-coupled ATP receptors, Ca²⁺ influx via receptor-activated TRPC5 channels elicits NO production from eNOS, which in turn induces secondary activation of TRPC5 channels via cysteine S-nitrosylation. TRPC5 is co-immunoprecipitated with eNOS and the scaffolding protein caveolin-1. Mutations in the caveolin-1-binding domains of TRPC5 impair their association and disrupt Ca²⁺ influx and NO production, suggesting that caveolin-1 is the scaffold that enables TRPC5 and eNOS to assemble into the signaling complex. Interestingly, ATP receptor-activated Ca²⁺ influx dissociates eNOS from caveolin-1 but enhances the association between TRPC5 and eNOS at the plasma membrane. This may relieve eNOS from negative regulation by caveolin-1 and promote the production of NO in the vicinity of TRPC5, leading to an efficient secondary activation of TRPC5 via S-nitrosylation. Thus, our study provides evidence that the TRPC5 channel-cavolin-1-eNOS complex dynamically coordinates NO and Ca²⁺ signaling in vascular endothelial cells.

The blood-brain barrier (BBB) is a physical barrier that limits substance transfer to the brain and maintains the homeostasis of the central nervous system. The breakdown of BBB leads to hypoxic encephalopathy, cerebral infarction, and neurodegenerative diseases. Cerebral ischemia, such as cerebral infarction, causes cell damage from hypoxia. However, it remains unclear how hypoxia affects brain capillary endothelial cell (BCEC) functions. Therefore, we focused on the Ca²⁺-activated Cl^- (Cl_Ca) channels, TMEM16A, in an immortalized bovine brain endothelial t-BBEC117 cells. Under hypoxic conditions, the expressions of mRNA and protein of TMEM16A channels in t-BBEC117 cells were upregulated and the activity of Cl_Ca currents were increased. Hypoxic stress enhanced cell proliferation and the enhancement was significantly suppressed by 100 µM niflumic acid or TMEM16A siRNA. The expression of HIF-1a protein downregulated under acute hypoxic conditions. In conclusion, the expression level of TMEM16A is increased and the cell proliferation is enhanced due to hypoxic stress. These findings suggest that TMEM16A channels are involved in the BBB functions during cerebral ischemia.

Background: Doxorubicin (DOX) is an anticancer drug used in the treatment ofvarious malignant tumors. DOX has side effect of cardiotoxicity and developmentof preventive drug is necessary. Until now, no effective drug was discovered.One of proposing mechanism to reverse cardiotoxicity is trough reversingmitochondrial dysfunction caused by calmodulin kinase II (CamKII).

Objective: To establish molecular basis for the development of prophylacticagents for DOX-induced cardiotoxicity focusing on CamKII-Mitochondriapathway.

Method: DOX treatment was performed in H9C2 cells. Mitochondrial membranepotential (MMP) detected using JC-1 dye, cell that undergo mitophagy weredetected by using Mitophagy detection dye. A specific inhibitor of CamKIIactivity, KN93 was used. we still prospecting result from western-blot andexpression level.

Results: Doxorubicin-induced MMP damage was significantly suppressed by KN-93treatment (red / green fluorescence signal ratio: control 3.1 ± 0.2, DOX 2.0 ±0.3, KN-93 group 2.4 ± 0.5, P value <0.05, n = 12). Similarly, DOX-inducedmitophagy was significantly suppressed by KN-93 treatment (mitophagy positivecells: control 58.0 ± 4.0%, DOX 58.0 ± 4.0%, KN-93 group 39.5 ± 8%, P value<0.05, n = 4).

Conclusion: DOX activates the CamKII and Drp-1 proteins and causes mitochondrialdysfunction due to mitochondrial injury and abnormal division of mitochondria.Molecules involved in the activation of CamKII and its signalling pathway aretarget molecules for the development of prophylactic agents for DOX-inducedcardiotoxicity

Abstract: The greatest advantage of iPS cell technology is that undifferentiatedcells can be obtained from the patient's own cells. Although iPS celldifferentiation is easy using the established cell line, its efficiency isextremely poor using patient-derived primary cultured cells because ofpathological aging. The purpose of this study was to establish a highlyefficient iPS induction method that can be applied to poor quality somatic cellssuch as pathogenic cells and senescent cells. Method and material: Dermalfibroblasts were isolated and cultured from three elderly patients who hadundergone cardiac surgery. As next step, episomatic vectors containing the fourYamanaka factors were transformed into these cells to induce iPS cells. Since itis difficult to induce iPS from pathogenic and senescent cells using thestandard iPS cell protocol, we had developed the optimal induction method. Then,we would verify whether the established iPS cell induction method usingfour-chamber myocardial tissue and fibroblasts derived from dermal tissue ofpatients with end-stage heart failure isolated by heart transplantation aspathological somatic cells. Results and conclusion: Pathological fibroblasts aremainly myofibroblasts that highly express alpha-smooth muscle actin and theywere factors that attenuated iPS induction efficiency. Therefore, by controllingthe factors in the culture medium, we succeeded in inducing iPS cells frompathological senescent cells obtained from the heart tissue of patient withend-stage heart failure.

Lacrimal fluid (tears) is important for preservation of the ocular surface, and thus dry eye induced by lacrimal hyposecretion in Sjögren's syndrome (SS) leads to reduced quality of life. However, the cause of lacrimal hyposecretion remains unknown, even though many studies have been conducted from the perspective of inflammation. Here, we hypothesized that a non-inflammatory factor induces lacrimal hyposecretion in SS pathophysiology. To elucidate such a factor, we conducted transcriptome analysis of the lacrimal glands in male non-obese diabetic (NOD) mice as a SS model. The result revealed that only four genes, including arginase 1, were downregulated in the lacrimal glands of male NOD mice after onset of lacrimal hyposecretion and dacryoadenitis. Furthermore, non-dacryoadenitis-type NOD mice were used to investigate the relationships among arginase 1 expression, lacrimal hyposecretion and dacryoadenitis. Non-dacryoadenitis-type NOD mice showed reduced tear secretion and low expression level of arginase 1. In addition, in BALB/c mice, an arginase 1 inhibitor reduced tear secretion. In conclusion, a non-inflammatory factor, arginase 1, is involved in lacrimal hyposecretion in male NOD mice, regardless of dacryoadenitis status. These results shed light on the pathophysiological role of arginase 1 in SS (dry eye).

Successful tissue regeneration requires a spatiotemporally integrated tissue organization by cell groups of various lineages. Therefore, it is important to evaluate tissue-supportive materials in terms of their feasibility to support the tissue organization. Recently, it has been noted that macrophages play important roles in the healing and regeneration process of damaged tissues through their reciprocal activation into inflammatory M1 and anti-inflammatory M2 subtypes. This study took a cynomolgus monkey as a primate model animal for better translational study. Pulpectomy was conducted in cynomolgus monkey tooth under general anesthesia, and then, different pulp capping materials were applied. We analyzed pulp regeneration process using immunohistochemistry-based quantitative fluorescence imaging. There was a correlation between the morphological dynamics of tissue macrophages and the success or failure of tissue regeneration. Our data suggested that regulation of spatiotemporal macrophage dynamics could be a key factor for proper dentin and tooth pulp regeneration in primates. Conclusively, our quantitative fluorescence imaging analysis of macrophage dynamics could provide a valuable method for evaluating materials and drugs supporting tissue regeneration.

Teriparatide (TPTD) is an anti-osteoporotic human PTH analogue that improves bone mass and biochemical properties of bone collagen deeply involved in the strength and quality of bone. However, visualization and quantification of topological properties of bone collagen have long been expected as a diagnostic approach for bone quality, but not well established. This study established a novel combinatorial approach of advanced nonlinear optics imaging (SHG: Second Harmonic Generation) and AI-based automatic morphometrical recognition method.

We applied cynomolgus monkeys as a model animal to fill the critical gap between human and conventional rodent models in terms of bone metabolism and its pharmacological evaluation. TPTD (low dose group: 1.2 µg /kg, high dose group: 6.0 µg /kg) was administered once a week for 18 months to ovariectomized monkeys as an osteoporosis model.

After the TPTD administration for 18 months, the 7th lumbar vertebrae of cynomolgus monkeys were dissected and analyzed by bone morphometry and quantitative SHG imaging.

In the secondary trabeculae of vertebral bone, where remodeling was active, the linearity of collagen fibers and bundles was decreased by OVX, however, recovered by TPTD administration in a dose-dependent manner.

This analysis enables us to visualize and quantify the topological properties of bone collagen fibers and bundles. Moreover, not only the biochemical properties of collagen but the topological properties of collagen fibers contribute to improving the osteogenic effect of TPTD.

In chondrocytes, changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) modulate chondrogenesis, cell proliferation, and death. Enhanced Ca²⁺ signaling facilitates the production of osteoarthritis (OA) markers including pro-inflammatory mediator (TNF-α) and matrix degrading enzymes (ADAMTS5 and MMP-13). However, the molecular mechanism responsible for the abnormal Ca²⁺ signaling is unknown. Here, we examined the functional roles of Ca²⁺ release-activated Ca²⁺ (CRAC) channels on Ca²⁺ signaling and OA marker production evoked by interleukin (IL)-1β, a major cytokine associated with OA, in mouse primary-cultured chondrocytes. IL-1β (5~10 ng/ml, for 2~6 h) upregulated the expression of OA markers and increased the number of [Ca²⁺]_i oscillation-positive chondrocytes. Furthermore, IL-1β facilitated NFAT translocation into nucleus. A selective CRAC channel inhibitor, 10 mM YM58483, significantly attenuated IL-1β-induced [Ca²⁺]_i oscillation, NFAT translocation, and OA marker production. A calcineurin inhibitor, 1 mM cyclosporin A, blocked both NFAT translocation and OA marker expression. These results suggest that IL-1β enhances Ca²⁺ influx through CRAC channels and activates calcineurin-NFAT signaling, and thus, upregulated the expression of OA markers. CRAC channels may be a therapeutic target for the treatment of OA.

The dynamics of voltage-dependent Ca²⁺ channel (VDCC) complexes are crucial for neuronal excitability, neurotransmitter release, and neuronal morphology. Recently, VDCC beta-anchoring and -regulatory protein (BARP) was identified as a novel regulator of VDCC activity via the interaction with VDCC β subunits. However, its physiological significance has been largely unknown. In this study, we reveal that BARP regulates the dynamics of VDCC complexes at dendritic spine. Fluorescence recovery after photobleaching assay revealed that BARP accelerates the fluorescence recovery of both VDCC α₁ and β subunits at dendritic spine. BARP forms VDCC α₁/β/BARP complex via BARP Region II and this ternary complex is important for accelerating the dynamics of VDCC complex. Cell surface labeling assay showed that BARP reduces the expression level of VDCC α1 subunit at the plasma membrane. Pharmacological studies suggest that BARP decreases the insertion of VDCC complex into the plasma membrane. These results suggest that BARP regulates the Ca²⁺ signaling at dendritic spine by altering the dynamics of VDCC complexes.

Whole-cell patch-clamp recordings were made from striatal ChINs in young-adult mice (P40-51) brain slices. ChINs were voltage-clamped at −60 mV. We used transgenic mice which expressing channelrhodopsin-2 in the striatal MSNs to selectively stimulate these neurons. Light stimulation evoked GABA_A receptor-mediated inhibitory postsynaptic currents (IPSCs) in the presence of glutamate and glycine receptor antagonists. A muscarinic acetylcholine receptor agonist, carbachol, inhibited IPSCs (1 μM: by 33.2 ± 4.4%, n = 9). To examine the changes in GABA release probability, we calculated coefficient of variation (CV), an index of change in release probability before and after application of 1 μM carbachol. Carbachol significantly increased CV from 0.21 ± 0.02 to 0.27 ± 0., suggesting that GABA release probability was changed by carbachol. The inhibitory effect of 1 μM carbachol was reduced to 0.22 ± 4.2% in the presence of pirenzepine, a selective M1 receptor antagonist (n = 6). However, pre-application of a selective M2 receptor antagonist, AF-DX 116 100 nM, did not reduce the inhibitory effect of carbachol (by 40.8 ± 5.0%, n = 8). These results suggest that activation of presynaptic M1 receptor inhibits GABA release from MSNs onto ChINs.

Renin-angiotensin system is believed to have important roles in blood pressure regulation. Baro and chemoreceptor afferents project to the nucleus tractus solitarius (NTS) neurons. The expression of angiotensin II type 1 (AT₁) and 2 (AT₂) receptors in the NTS is confirmed. However the physiological roles of angiotensin II (Ang II) in NTS are not fully understood. We have previously reported that ang II increases the frequency of spontaneous EPSCs (sEPSCs) through the activation of AT₁ receptors and decreases it through AT₂ receptor activation. Contrary, Ang II augmented the amplitude of tractus solitarius evoked EPSCs (eEPSCs) via the activation of AT₂ receptors and decreased it by AT₁ receptor activation. In this report we aimed to reveal the involvement of NO cascade in these effects of Ang II by using a slice patch-clamp technique.

 The prior application of L-NAME inhibited the excitatory effects of Ang II on sEPSCs via AT₁ receptors but not antagonized the inhibitory effects of on sEPSCs via AT₂ receptors. Similarly, prior application of L-NAME antagonized the inhibitory effects of Ang II on eEPSCs via AT₁ receptors but not antagonized the augmenting effects on eEPSCs by AT₂ receptors.

These results suggest that the activation of AT₁ receptors in the NTS induces NO production and produced NO induces facilitation of sEPSCs and inhibition of eEPSCs.

Synaptic vesicle glycoprotein 2A (SV2A) is specifically expressed in synaptic vesicles and regulates neurotransmitter release at synapses. Recent studies reported a significant association of genetic SV2A polymorphism with schizophrenia (Schizophr. Res., 141, 262, 2012). We performed behavioral study using the SV2A-mutant rats, which carry a loss-of-function mutation (L174Q) in the Sv2a gene, to elucidate the effect of levetiracetam (LEV, a SV2A ligand) on methamphetamine (MAP)-induced hyperactivity in Sv2a^L174Q rats. Acute treatments with MAP (1 mg/kg, i.p.) significantly increased locomotor activities in Sv2a^L174Q rats, but not in control (F344) rats. The MAP-induced hyperactivity in Sv2a^L174Q rats was weakly potentiated by LEV (30 mg/kg, i.p.). In F344 rats, LEV markedly potentiated MAP-induced hyperactivity to the extent similar to Sv2a^L174Q rats. In addition, D₂ blocker, haloperidol (0.3 mg/kg, i.p.) completely suppressed the augmentation of MAP-induced hyperactivity by LEV in both Sv2a^L174Q and F344 rats. The present results suggest that dysfunction of SV2A induces behavioral supersensitivity to MAP by increasing dopamine release, which may be linked to vulnerability against schizophrenia. Further, LEV reduces the function of SV2A, suggesting that LEV may exacerbate psychiatric symptoms.

The rhomboid nucleus of the bed nucleus of the stria terminalis (BSTrh) is one of the important brain regions for infanticide. We previously revealed that excitatory synaptic transmission in the BSTrh is potentiated in adult male mice compared with those before sexual maturation. However, it remains unclear how sexual hormone mediates this behavioral transition and plastic changes in the neural circuit underlying behavioral selection for pups during sexual maturation. Here, we found that continuous administration of estradiol (E2) to gonadectomized mice during sexual maturation elicited infanticidal behaviors in adulthood (about 7 weeks old). Next, we performed whole-cell patch clamp recording in the BSTrh to study the effect of altered gonadal steroid hormones levels on neural synaptic transmission. We found that E2 administration considerably enhanced excitatory synaptic transmission in the BSTrh by increasing the probability of glutamate release from the presynaptic terminals. These data suggest that reinforcement of excitatory synaptic transmission by estrogen-receptor-dependent signaling in the BSTrh during sexual maturation may contribute to the expression of infanticidal behaviors. We currently explore the source of excitatory synaptic inputs into the BSTrh.

Identification and characterization of the molecules that regulate neurite outgrowth are essential for understanding how brain circuits form and function. In this study, we applied 10 different phospholipids to cultured cortical neurons and found that lysophosphatidylethanolamines (LPEs) with different fatty acid lengths, palmitoyl LPE (16:0 LPE) and stearoyl LPE (18:0 LPE), stimulate neurite growth in cultured cortical neurons. Noteworthy, inhibitor of Gq/11 protein inhibited 16:0 LPE-stimulated neurite outgrowth but not 18:0 LPE-stimulated neurite outgrowth. In contrast, inhibitor of Gi/Go proteins inhibited 18:0 LPE-stimulated neurite outgrowth but not 16:0 LPE-stimulated neurite outgrowth. The effects of PKC inhibitors on neurite outgrowth were different between 16:0 LPE- and 18:0 LPE-treated cultures. We also found that both 16:0 LPE and 18:0 LPE activate MAPK to the same extent. MAPK inhibitor completely inhibited 18:0 LPE-stimulated neurite outgrowth and partially inhibited 16:0 LPE-stimulated neurite outgrowth. These results suggest that 16:0 LPE and 18:0 LPE stimulate neurite outgrowth through distinct signaling cascades in cultured cortical neurons and that distinct G protein-coupled receptors are involved in these processes.

Polyunsaturated fatty acids (PUFAs) are essential for brain development and function. Increasing evidence has shown that an imbalance of PUFAs is associated with various human psychiatric disorders, including autism and schizophrenia. Fatty acid-binding proteins (FABPs), cellular chaperones of PUFAs, are involved in their intracellular trafficking, signal transduction, and gene transcription. Previously, we showed that FABP3 is strongly expressed in the parvalbumin-expressing interneurons (PV neurons) of the mouse anterior cingulate cortex (ACC), which is a component of the limbic cortex and is important for the coordination of cognitive and emotional behaviors. In addition, FABP3 regulates GABA synthesis through transcriptional regulation of Gad67 in the ACC and that methionine restores normal Gad67 expression and behaviors in Fabp3 knockout (KO) mice. In this study, we analyzed the density and the percentage of PV neurons surrounded by perineuronal nets (PNNs) in the ACC of adult Fabp3 KO mice. PNNs are key components of extracellular matrix that enwrapping PV neurons and regulate synaptic plasticity. PV density increased in the ACC of adult Fabp3 KO mice, whereas the number of PV-neurons remained unchanged. The density of PNN and the number of PNN-positive PV neurons were significantly increased in the ACC of adult Fabp3 KO mice. These findings demonstrate that FABP3 is involved in the control of expression of PV and formation of PNNs in the ACC, thus suggesting the importance of PUFA homeostasis in the ACC for maturation of PV neurons.

The serotonin transporter (SERT) is responsible for the termination of serotonergic neural transmission via reuptake of serotonin (5-HT) at nerve terminals. We investigated the involvement of the ubiquitin E3 ligase HRD1 (HMG-CoA reductase degradation protein), which participates in endoplasmic reticulum (ER)-associated degradation (ERAD), in the functional regulation of SERT in order to elucidate the regulation of SERT via its membrane trafficking, We used HEK293 cells and COS7 cells transiently expressing wild-type SERT or a SERT C-terminal deletion mutant (SERTΔCT), a SERT protein predicted to be misfolded. Studies using HRD1-overexpressing or HRD1-knockdown cells demonstrated that HRD1 is involved in SERT proteolysis. Overexpression of HRD1 promoted SERT ubiquitination, the effect of which was augmented by treatment with the proteasome inhibitor MG132. Immunoprecipitation studies revealed that HRD1 interacts with SERT in the presence of MG132. In addition, HRD1 was intracellularly colocalized with SERT, especially with aggregates of SERTΔCT in the ER. HRD1 also affected SERT uptake activity in accordance with the expression levels of the SERT protein. These results suggest that HRD1 contributes to the membrane trafficking and functional regulation of SERT through its involvement in ERAD-mediated SERT degradation.

Nicotine excites the inferior olive (IO) neurons and induces kinetic tremor via activating α7 nACh receptors. Meanwhile, nicotine is also known to enhance dopamine release by activating presynaptic nACh receptors, but the role of dopaminergic system in regulating nicotine-induced tremor remains unknown. Here, we investigated the effect of various dopamine receptor antagonists on nicotine-induced tremor in mice and rats. Animals were pretreated with dopamine antagonists 15 min before nicotine (1 mg/kg) injection. Nicotine tremor was significantly facilitated by the selective D_1/5 antagonist SCH-23390 while it was significantly inhibited by the D₃ selective antagonist U-99194. Neither D₂ (L-741,626) nor D₄ antagonist (L-745,870) affected nicotine tremor. In addition, microinjection of SCH-23390 into the IO tended to enhance nicotine tremor, but its injection into the striatum showed no effect. Microinjection of U-99194 into the IO or the striatum did not change nicotine tremor, however, its injection into the cerebellum significantly attenuated nicotine tremor. These results suggest that the dopaminergic system regulates nicotine-induced tremor biphasically, D₃ receptors in the cerebellum being facilitatory and D_1/5 receptors in the IO inhibitory.

Unlike intracellular Ca²⁺, extracellular Ca²⁺ ([Ca²⁺]_o) has received only limited attention. [Ca²⁺]_o has profound effects on neuronal membrane potential and synaptic transmission. Physiological conditions like sensory stimulation, sleep-wake transitions and pathological conditions like seizures, ischemia are reported to decrease [Ca²⁺]_o. However, these data are obtained from a single point analysis using a Ca²⁺ electrode and lack spatiotemporal information of [Ca²⁺]_o changes. To understand processes shaping local and global [Ca²⁺]_o change involved in regulating physiological and pathological neuronal activity, we need an imaging technique that provides higher spatiotemporal resolution. Thus, we developed a label-free [Ca²⁺]_o image sensor (CIS) with a time resolution of a few tens of milliseconds at 23.55 mm pitch of 128 x 128 pixel. The CIS is highly selective to Ca²⁺ but not to other cations within wide dynamic range (from 100 mM to 100 mM). We used CIS for the imaging of [Ca²⁺]_o in acute hippocampal slices. Stimulation with neurotransmitter glutamate (Glu), decreased [Ca2+]o to around 800 mM within 10 s, which gradually returned to the baseline level (2 mM) with slow kinetics (5 min). The Glu-evoked [Ca²⁺]_o decrease was higher in CA1 and DG compared to CA3, and was inhibited by a NMDA receptor antagonist D-APV but not AMPA receptor antagonist CNQX. NMDA mimicked the Glu-evoked [Ca²⁺]_o decrease and, again was inhibited by D-APV. These findings suggest that Glu acts on neuronal NDMA receptors to reduce [Ca²⁺]_o with different spatiotemporal patterns, reflecting differences in NMDA receptors distribution. Our study demonstrates that a CIS can detect [Ca²⁺]_o changes associated with biological events and have broad applicability in future biological studies.

The hiPSC-derived neural networks on dish are expected to improve the predictability of CNS adverse effects at the non-clinical stage in drug development. In human brain, EAATs are responsible for removal of an excess amount of L-glutamate (L-Glu) and prevent neuronal damage caused by excitotoxicity. In this study, we studied the role of EAATs in the hiPSC-derived neural networks.

Commercially-available hiPSC-derived neurons were seeded at 3.0×10⁵ cells/cm². At 63 DIV, the synchronized burst firing activity, which was indicative of network formation, were confirmed by microelectrode array system. Synapsin1 puncta were adjacent to PSD95 puncta, supporting synaptic maturation in these neural networks. When 100 µM L-Glu was applied exogenously at 63 DIV, L-Glu concentration in the medium ([L-Glu]_o) became almost zero in 1 hr and cell viability was not affected. In the presence of TFB-TBOA (TFB), a non-specific EAATs inhibitor, [L-Glu]_o was not changed at all and the cell viability was severely decreased. When AP5, an NMDAR antagonist, was co-applied with TFB, the cells were significantly rescued. These results indicate that EAATs are functional in the hiPSC-derived neural networks and protect neurons from excitotoxicity. It is also suggested that hiPSC-derived neural networks can be applied for the development EAAT-related drugs.

In the multicellular system, a variety of signaling molecules are supposed to mediate cell-to-cell communication. However, the information on their spatio-temporal dynamics remains largely elusive, likely due to the lack of the suitable technology. Since fluorescence imaging is a powerful approach to monitor the dynamics of signaling molecules, I decided to develop fluorescent sensors for various signaling molecules.　First, I developed a green fluorescent sensor for oxytocin, a neuropeptide that play critical roles in social behaviors, food intake, and stress responses. Through the screening of hundreds of mutants, I finally obtained a sensitive fluorescent sensor that show a large fluorescence change (up to 700% dF/F0). I applied this sensor to in vivo fiber photometry measurement, and found the active oxytocin dynamics in the brain upon a various physiological stimuli. Furthermore, I tried to extend the repertoire of the target signaling molecule of fluorescent sensors by using a similar way to the development of the oxytocin sensor. Through the screening, I succeeded in obtaining sensitive green fluorescent sensor for ~20 signaling molecules including chemical transmitters, peptide ligands, and lipid mediators. These fluorescent sensors will further extend our knowledge on the intercellular signaling dynamics in living systems.

The pathological hallmark of Parkinson disease is a widespread distribution of the aggregated α-synuclein (α-Syn) proteins in the inclusions known as Lewy bodies. This widespread distribution of α-Syn oligomer is involved in intercellular transport between neurons and glial cells. However, it is unclear whether other blood-brain barrier (BBB) comprising cell types (brain endothelial cells, pericytes and astrocytes) contribute this process. Here, we investigated the ability of BBB comprising cell types, especially brain pericytes to take up α-Syn oligomer. We confirmed that HiLyte488-conjugated α-Syn (AnaSpec Inc.) contained monomeric (17 kDa) and oligomeric (> 17 kDa) α-Syn by Western blot. Oligomeric α-Syn uptake by pericytes at 1 hr was decreased during a 24-h period, but other cell types did not show a similar reduction. In the presence of a lysosome inhibitor bafilomycin A1 or P-glycoprotein (P-gp) inhibitor cyclosporin A (CsA), the intracellular accumulation of oligomeric α-Syn in pericytes was increased. In addition, CsA-induced increase of intracellular oligomeric α-Syn was degraded at 48 h. These results suggest that oligomeric α-Syn is degraded in pericytes and P-gp is involved in the efflux of oligomeric α-Syn from pericytes. The inhibition of P-gp by CsA would facilitate the degradation of oligomeric α-Syn by pericytes.

Brain pericytes located in the brain capillaries are surrounded by astrocytic endfeets where glutamate transporters express. The uptake of glutamate, an excitatory neurotransmitter, in astrocytes is mediated by sodium-dependent glutamate transporter including glial glutamate transporter (GLT)-1 to regulate the extracellular glutamate concentration. However, little is known about the role of pericyte on the glutamate homeostasis maintained by astrocyte. To evaluate an effect of the pericytes on glutamate uptake by astrocytes, we prepared human brain-derived pericyte and astrocyte coculture system. The uptake of [3H]-L-glutamate by astrocytes was expressed as the cell/medium ratio. The uptake levels of glutamate in astrocytes cocultured with pericytes were significantly higher than those in astrocyte monoculture. Elevated glutamate uptake in the astrocytes cocultured with pericytes was not observed under the condition with Na⁺-free medium and blocked by the selective GLT-1 inhibitor dihydrokainic acid. These data showed that pericytes increase glutamate uptake in astrocytes by upregulating transporter activity. Based on these findings, pericytes may facilitate glutamate clearance in brain pericapillary location by increasing the astrocytic glutamate uptake through sodium-dependent transporter including GLT-1.

The onset and progression of Alzheimer's disease (AD) correlate with neuroinflammatory processes, and inflammatory microglia (MG) are associated with AD-like pathology in a transgenic mouse model. GPNMB was identified as a marker for one subtype (type 1) of rat primary MG (Kawahara et al., GLIA, 2016). However, the function of GPNMB+ type 1 MG in AD brain are largely unknown. We recently reported that memory deficits in 9 month-old APP23 mice, one of the amyloid precursor protein transgenic mice, was reduced in Gpnmb gene heterozygosity (APP23;Gpnmb+/-, 92^ndAnnual meeting of the Japanese Pharmacological Society, 2-O-04, 2019). In the present study, we investigated whether Gpnmb gene heterozygosity could affect on memory deficits in 9-month old 5xFAD mice, other AD model mice. We used 5xFAD mice backcrossed to C57BL/6J mice at least for ten generation. Spatial learning and memory was evaluated by Morris Water Maze test. We observed that the number of Nissl-positive neurons in subiculum of 5xFAD mice were lower than those in their wild type littermates. We observed that AD-related memory deficits in 9 month-old 5xFAD mice were decreased in Gpnmb gene heterozygosity (5xFAD;Gpnmb+/-). These finding suggest that GPNMB-positive type 1 MG may be related to memory deficits in AD.

Cholesterol is one of the most abundant molecules that constitutes the plasma membrane. The total cholesterol content in the brain is over 20%, and 50% of myelin is made up of cholesterol. In several neurodegenerative diseases, accumulation of free cholesterol in the extracellular space due to demyelination and neuronal death is suggested. However, the effect of free cholesterol in the brain parenchyma have not been focused. We found that cholesterol induces calcium oscillation in cultured astrocytes but not in neurons. The calcium oscillation lasted at least 4 hours. Removal of extracellular calcium decreased the oscillation frequency, but not totally blocked, while inhibitors against IP₃ receptor, calcium -ATPase and PLC blocked the calcium oscillation. Therefore, cholesterol induces calcium oscillation in astrocytes through the PLC-IP₃ pathway. Cholesterol treatment for 24 hours led cell death in neurons but not in astrocytes. The survival rate of astrocytes under cholesterol decreased when the calcium oscillation was suppressed. These results suggest that the calcium oscillation induced by cholesterol helps astrocytes to survive in high extracellular cholesterol condition, which may imply an astrocytic behavior under pathological conditions where extracellular cholesterol concentration is raised.

[AIM] Extracellular zinc aggravates microglial M1 phenotype. Here, we investigated whether zinc-aggravated M1 microglia affects the astrocytic engulfing activity.

[METHODS] Mouse astrocytes were incubated with microglial-conditioned medium (MCM) collected from M1 microglia induced by lipopolysaccharide (LPS) after ZnCl₂ treatment. Astrocytic engulfment activity was examined by beads uptake assay. The mRNA level of P2X7 receptors (P2X7R) in astrocytes was measured by real-time PCR. Effect of A43809, a P2X7R antagonist, on MCM-induced astrocytic engulfment was also examined.

[RESULTS] MCM from M1 microglia increased the bead uptake by astrocytes. This increased engulfment activity was suppressed when astrocytes were treated with MCM from LPS-induced M1 microglia pretreated with ZnCl₂. P2X7R mRNA level was increased in astrocytes treated with MCM from M1 microglia, but not from M1 microglia pretreated with ZnCl₂. Treatment of astrocytes with A438079 resulted in attenuation of increase in the engulfment activity in astrocytes after MCM treatment.

[CONCLUSION] These results suggest zinc pretreatment abolishes the ability of M1 microglia to increase the engulfing activity in astrocytes via alteration of astrocytic P2X7R.

Prostaglandin E₂ (PGE₂) plays an important role in modulating microglial function. In the present study, we have found that PGE2 increases protein of cyclooxygenase-2 (COX-2), which are involved in PGE₂ synthase in cultured rat microglia.

COX-2 protein was increased by PGE₂ at 10^-6 M for 3 h in microglia. The increase was inhibited by PF-04418948 (EP₂ antagonist), but not by ONO-8713 (EP₁ antagonist), ONO-AE3-240 (EP₃ antagonist), or ONO-AE3-208 (EP₄ antagonist) at 10^-6 M. In addition, ONO-AE1-259-01 (EP₂ agonist), also increased COX-2 protein in a dose dependent manner, and this protein level was not affected by ONO-DI-004 (EP₁ agonist), ONO-AE-248 (EP₃ agonist), or ONO-AE1-329 (EP₄ agonist) at 10^-6 M. Moreover, PGE₂ at 10^-6 M for 3 h had little effect on the protein expression of cyclooxygenase-1, microsomal prostaglandin E synthase-1, microsomal prostaglandin E synthase-2, or cytosolic prostaglandin E synthase, which are also involved in PGE₂ synthase.

Therefore, activation of EP₂ receptor results in the increase of COX-2 protein in microglia.