A stimulation inducing long-term potentiation (LTP) of synaptic transmission induces a persistent expansion of dendritic spines, phenomena recognized as structural LTP (sLTP). Actin cytoskeleton plays an essential role in this process. We previously proposed the persistent interaction between CaMKII and Tiam1, an activator of Rac is a critical mediator of actin regulation. Interestingly, the formation of the complex locks CaMKII into an active conformation, which in turn maintains the activity of Tiam1 though phosphorylation. This makes Rac1 activity persist in a stimulated spine. Therefore, the CaMKII-Tiam1 complex plays a pivotal role in sLTP.

To understand the significance of the CaMKII-Tiam1 complex in vivo, we generated Tiam1 knocked-in mice where critical residues for CaMKII binding were mutated into alanines. In the KI brain, Rac1 activity was specifically reduced compared with WT littermate. Also sLTP was abolished in hippocampal neurons from KI. Gross appearance of brain structure and spine density was normal in KI mice. The KI mice showed decreased object recognition memory 7 days after training while the other behavioral tests were normal. Thus, the CaMKII-Tiam1 interaction is not only crucial for sLTP but also requires for memory storage.

The aim of the present study was to examine enhancement of learning and memory by oral administration of ergothioneine (ERGO), which is a hydrophilic antioxidant highly contained in golden oyster mushrooms and other foods, and systemically absorbed by its specific transporter OCTN1/SLC22A4 in daily life, with an aim to clarify its possible role as a neurotropic compound. After oral administration of ERGO in normal mice, the novel object test revealed a longer exploration time for the novel object than for the familiar object. Similar result was also confirmed in mice ingested with ERGO-free diet. Dietary-derived ERGO is present in the body without the administration, but the ERGO administration led to modest (3～4 times) increase in its concentration in plasma and hippocampus. Exposure of cultured hippocampal neurons to ERGO elevated the expression of the synapse formation marker, synapsin I, and neurotrophin-3 and -5. The elevation of synapsin I was inhibited by tropomyosin receptor kinase inhibitor K252a. Thus, oral intake of ERGO may enhance object recognition memory, and this could occur at least partially through promotion of neuronal maturation in the hippocampus.

Obsessive-compulsive disorder (OCD) is a psychiatric disorder characterized by repetitive inappropriate thoughts (obsessions) and behaviors to get rid of obsessions (compulsions). Although selective serotonin reuptake inhibitors (SSRIs) are the first-choice treatment for OCD, response rates to SSRI treatment vary between symptom dimensions. In this study, to find a therapeutic target for SSRI-resilient OCD symptoms, we evaluated treatment responses of quinpirole sensitization-induced OCD-related behaviors. Chronic SSRI administration rescued the cognitive inflexibility and the hyperactivity in the lateral orbitofrontal cortex (lOFC), while repetitive behavior was not improved by SSRI. D₂ receptor signaling in the central striatum (CS) was involved in SSRI-resistant repetitive behavior. An adenosine A_2A antagonist, istradefylline rescued abnormal excitatory synaptic function in the CS indirect pathway medium spiny neurons of sensitized mice and also alleviated both of the QNP-induced OCD-related behaviors with only short-term administration. These results provide a new insight into therapeutic strategies for SSRI-resistant OCD symptoms and indicate the potential of A_2A antagonists as a rapid-acting anti-OCD drug.

It is crucial for animals to select an appropriate behavior in a conflict situation where they face both positive and negative motivation. But the neural circuit mechanism underlying behavioral selection in a conflict situation is unclear. We set up a behavioral paradigm in which mice need to explore an experimental context where they might receive electric shocks in order to obtain sucrose solution. Using c-Fos expression as a marker of neuronal activation, we found that the anterior cingulate cortex (ACC) and periaqueductal gray (PAG) were activated in the conflict situation. Anterograde and retrograde tracing with AAV-CaMKII-eYFP and Fluoro-Gold, respectively, revealed dense projections from the ACC to the PAG. To record ACC-PAG activities from behaving mice, a genetically encoded calcium indicator, GCaMP6, was expressed selectively in the ACC-PAG pathway and its fluorescence was detected through an optic fiber. ACC-PAG activity was associated with sucrose-seeking behavior. Furthermore, optogenetic activation of the ACC-PAG pathway shortened the latency to obtain sucrose. These results suggest that ACC inputs to the PAG facilitate reward-seeking behavior in the conflict context.

The pathophysiological role of periostin (POSTN), expression of which is increased in right ventricles of monocrotaline (MCT)-induced pulmonary arterial hypertension (PAH) model rats, has not been clarified. We investigated the effect of POSTN on inducible nitric oxide synthase (iNOS) expression, which causes cardiac dysfunction, in right ventricular fibroblasts (RVFbs). PAH model rats were produced by an intraperitoneal injection with MCT (60 mg/kg). In RVFbs isolated from PAH model rats, the iNOS expression and phosphorylation of extracellular signal-regulated kinase (ERK) 1/2, c-Jun N-terminal kinase (JNK) and nuclear factor (NF)-kB were higher than RVFbs isolated from control rats. Recombinant POSTN increased iNOS expression and NO production in RVFbs isolated from normal rats, which were prevented by a pharmacological inhibition of ERK1/2, JNK or NF-kB. The culture medium of recombinant POSTN-stimulated RVFbs suppressed L-type Ca²⁺ channel (LTCC) activity in H9c2 cardiomyoblasts. We demonstrated that POSTN increased iNOS expression and subsequent NO production in RVFbs. The enhanced NO production in RVFbs might be associated with the right ventricular dysfunction via the suppression of LTCC activity of cardiomyocytes in PAH.

Canstatin, a cleaved C-terminal fragment of type IV collagen α2 chain, inhibits hypoxia-induced apoptosis in H9c2 cardiomyoblasts. After myocardial infarction, the expression of canstatin is decreased in the infarcted area. We investigated whether the canstatin-treatment exerts a cardioprotective effect on heart failure after myocardial infarction in rats. The myocardial infarction model rats induced by ligating left anterior descending artery were intraperitoneally injected with recombinant canstatin (20 µg/kg/day) or vehicle for 28 days after the operation. Echocardiographic analysis showed that canstatin-treatment significantly inhibited cardiac dysfunction and tended to inhibit left ventricular dilation. Canstatin did not change the ratio of infarcted area to left ventricular area. In the histological analysis of non-infarcted area, canstatin inhibited hypertrophy of cardiomyocytes (using hematoxylin and eosin staining) and interstitial fibrosis (using picro-sirius red staining). The present study for the first time demonstrated that chronic recombinant canstatin-treatment prevents heart failure after myocardial infarction through the inhibition of cardiac hypertrophy and fibrosis in non-infarcted area.

Background- We previously reported that 2,5-dimethylcelecoxib (DMC) activated glycogen synthase kinase-3 (GSK-3), a negative regulator of cardiac hypertrophy, in mice. In this study, we examined the effects of DMC on isoprenaline (ISO)-induced cardiac hypertrophy and fibrosis using in vivo and in vitro systems.

Methods- ISO (20 mg/kg/day) was administered to C57BL/6J mice using an osmotic pump, and DMC (1,000 ppm) was added into feed for 14 days. Mice were divided into 3 groups: Control, ISO and ISO+DMC. Primary neonatal rat ventricular cardiomyocytes (NRVCs) and adult rat cardiac fibroblasts (RCFs) were pretreated with DMC (3-20 mmol/L) from 1 hour before incubation with ISO (5 mmol/L) for 24 hours. NRVC sizes and RCF proliferation were measured and proteins were examined.

Results- DMC prevented hypertrophy and fibrosis in vivo. DMC attenuated the enlargement of NRVCs by activating GSK-3 and suppressing β-catenin and mTOR. DMC also attenuated RCF proliferation by activating GSK-3 and suppressing cyclin D1. The direct involvement of GSK-3 was verified using a GSK-3 inhibitor SB216763.

Conclusion- DMC prevented cardiac hypertrophy and fibrosis by activating GSK-3 and modulating downstream proteins.

Angiotensin-converting enzyme 2 (ACE2) is a negative regulator of renin-angiotensin system and has the beneficial effects on the cardiovascular diseases. We elucidate that the B38-CAP, a carboxypeptidase derived from Paenibacillus sp.B38, has ACE2-like enzymatic activity. In silico analysis revealed the structural similarity between B38-CAP and rhACE2 despite the lack of obvious sequence homology. In vitro recombinant B38-CAP protein catalyzed the conversion of angiotensin II to angiotensin 1-7 with the same potency as rhACE2. Treatment with B38-CAP reduced plasma angiotensin II levels and suppressed angiotensin II-induced hypertension, cardiac hypertrophy and fibrosis in mice. Moreover, continuous infusion of B38-CAP inhibited pressure overload-induced pathological hypertrophy, myocardial fibrosis, and cardiac dysfunction in mice. Importantly, B38-CAP treatment did not induce overt toxicity of liver and kidney. B38-CAP is an ACE2-like carboxypeptidase, which is functional in vitro and in vivo. B38-CAP could be a novel therapeutics in cardiovascular diseases.These results suggest that the strategy to find molecules of convergent evolution might be effective for drug development, such as ‘generic' protein preparation of functional enzymes.

【Background】

Runx2 is a critical regulator of osteoblast differentiation; however, the pathophysiological significance of Runx2 in cardiac homeostasis remains to be elucidated.

【Methods and Results】

Murine myocardial infarction (MI) was generated by ligating the left coronary artery. Quantitative RT-PCR and immunoblot analyses revealed the up-regulation of Runx2 mRNA and protein in post-infarct myocardium. Immunofluorescent staining and flow cytometric analyses showed that Runx2 was expressed in heart-infiltrating CD11b⁺ myeloid cells after MI. To analyze the biological functions of Runx2 during post-infarct cardiac remodeling, myeloid cell-specific Runx2 deficient mice (CKO mice) were exposed to MI. MI induced severe heart failure and lung congestion in CKO mice. CKO mice showed exacerbated cardiac fibrosis and function, respectively after MI. RNA sequence analyses demonstrated that myeloid expression of Runx2 regulated the gene expression responsible for vascular functions. Consistently, capillary density was decreased in CKO mice, proposing the importance of Runx2-expressing myeloid cells in cardiac repair. Single-cell RNA sequence analyses showed that Runx2-expressing cells did not coincide with either M1 and M2 macrophages.

【Conclusion】

Runx2-expressing myeloid cells prevented post-infarct cardiac remodeling as a novel cell population.

Intracellular Ca²⁺ signal controls various cell functions such as gene expression, cell death, proliferation, and migration. In immune cells, the activation of K⁺ channels promotes Ca²⁺ entry and therefore regulates cell functions. Inwardly rectifying K⁺ (Kir) channels play important roles in the formation of resting membrane potential in various types of cells. Kir2.1 channel is expressed in macrophages, however, the contribution of Kir2.1 channels to the pathophysiological functions is unclear. In this study, we examined the pathophysiological roles of Kir2.1 channels in murine bone marrow-derived macrophage (BMDM). The resting membrane potential in BMDM significantly depolarized by 100 μM Ba²⁺ (a Kir blocker). The resting [Ca²⁺]_i and store-operated Ca²⁺ entry (SOCE) induced by 1 μM thapsigargin were significantly reduced by 100 μM Ba²⁺. Neither application of Ba²⁺ nor ML133 (a Kir2 selective blocker) affected differentiation of myeloid progenitor cell into BMDM, phagocytosis, and proliferation. On the other hand, migration of BMDM was significantly inhibited by these Kir2.1 inhibitors. These results suggest that the activity of Kir2.1 channels regulates resting membrane potential and [Ca²⁺]_i, and thus promotes migration to inflammatory regions in macrophage.

The alkaline pH-activated K_2P5.1 K⁺ channel contributes to the setting of the resting potential and the control of Ca²⁺ signaling. K_2P5.1 is up-regulated in CD4⁺ T cells from patients of autoimmune diseases such as rheumatoid arthritis and multiple sclerosis. We have reported that K_2P5.1 is up-regulated in inflammatory CD4⁺ T (Th1) cells of inflammatory bowel disease (IBD) model mice and that K_2P5.1 is a possible therapeutic target for IBD. However, it remains unclear the mechanisms underlying up-regulation of K_2P5.1 in Th1 cells. We recently showed that Ca²⁺-activated K⁺ channel K_Ca3.1 was post-transcriptionally regulated by histone deacetylases (HDACs) in Th1 cells of IBD model mice. Present study showed no changes in the expression levels of K_2P5.1 by HDAC inhibitor treatment in CD4⁺ cells. Hypoxia underlies the polarization of the Th1 lymphocytes in inflamed tissues. Up-regulation of hypoxia-inducible factor (HIF)-1α was found in CD4⁺ T cells of IBD model mice. Hypoxia (1.5% O₂) for 24 hr increased the expression levels of K_2P5.1 in mice CD4⁺ thymocytes. These results suggest that K_2P5.1 may be up-regulated in Th1 cells of IBD model through HIF-1α-mediated signaling pathway.

Agonist induces substantial changes in a cytoplasmic tail of G-protein coupled receptors. However, structural consequences of such changes have not been detected in V1a receptors, which was stimulated by full agonist. Here, we explored receptor-receptor interaction by using NanoBit split luciferase system. One of two parts of split luciferase were connected to the carboxy-terminal tail of the V1a receptor. These modifications of V1a tails did not alter vasopressin-mediated intracellular calcium signals. When V1a receptors were connected with small or large fragment of nanoluciferase and were co-expressed, strong luminescent signal was detected, indicating that V1a receptor tails formed full nanolucuferase molecule. We found that signal intensities of luciferase light gradually increased during measurement period of five minutes in human embryonic kidney cells after substrate additions. Vasopressin significantly reduced the luciferase signal intensity during the measurement period. Our data indicate that split nanoluciferase system is useful to detect small conformational changes in the cytoplasmic tails of the agonist-stimulated V1a receptor homodimers.

Background: Boron neutron capture therapy is a radiotherapy utilizing the neutron capture reaction. The commonly used boron agent, p-borono-l-phenylalanine (BPA), is accumulated in tumors by amino acid transporters upregulated in tumors. In this study, to propose a novel strategy of selective boron delivery targeting peptide transporter, we developed BPA-containing dipeptides (BPA-Tyr and Tyr-BPA) and examined their interaction with peptide transporters and their uptake into tumor cells. Methods: We established HEK293 cells stably expressing PEPT1 or PEPT2, and examined their interaction with BPA-Tyr and Tyr-BPA. Dipeptide transport activity was compared among tumors with varied PEPT1 and PEPT2 expression levels. We evaluated the boron accumulation in tumors after the treatment of BPA-Tyr and Tyr-BPA in vitro and in vivo. Results: BPA-Tyr and Tyr-BPA are transported by PEPT1 and PEPT2. The peptide transport activity in tumor cells correlated with PEPT1 expression level. BPA-Tyr and Tyr-BPA were delivered into PEPT1-expressing tumor cells via a PEPT1-mediated mechanism in vitro. In vivo, intravenous administration of BPA-Tyr resulted in a higher accumulation in the tumors compared with the blood. Conclusion: PEPT1 is a promising target for cancer-specific boron delivery in BNCT, using BPA-containing dipeptide-based boron agents.

Retinal pigment epithelium (RPE) has an important role for maintenance of visual function by phagocytosis of photoreceptor outer segment. Recent studies have elucidated that this phagocytosis shares a non-canonical form of autophagic degradation. Also, patients with non-exudative age-related macular degeneration show dysregulation of autophagic degradation in RPE. To find effective therapeutic agent, we used in vitro photoreceptor outer segment phagocytosis assay by RPE cells. We found that an Nrf2 activating triterpenoid, RS9, accelerated photoreceptor outer segment phagocytosis. Notably, RS9 activated both AMPK/mTOR and Nrf2/SQSTM1 signaling pathway. These signals are known to facilitate autophagic degradation by different mechanism. In the early phase, RS9 phosphorylated AMPK, then dephosphorylated mTOR. In the late phase, RS9 activated Nrf2, then induced SQSTM1. A canonical Nrf2 activator diethyl maleate showed same effects; however, it has a much weaker potential compared with RS9. In conclusion, these findings indicate that RS9 is a prominent autophagy inducer and promising therapeutic agent against non-exudative age-related macular degeneration.

Acetylcholine is a critical neuromodulator for aversive learning and cognitive memory. For its association with Alzheimer's disease, it has drawn much of attention as the therapeutic target. The therapeutic drug improves learning and memory deficits in Alzheimer's disease, but unable to cure. Muscarinic receptor M1 (M1R) has been implicated in aversive learning and cognitive memory through its downstream mediator protein kinase C (PKC) activation. However, the mode of action of acetylcholine in neurons has not been clarified yet, as PKC signaling pathway remains unknown. Based on that acetylcholine level is the highest in striatum/nucleus accumbens (NAc) where it impacts emotional behavior, in this study we aimed to clarify the M1R-PKC pathway in striatum/NAc leading to aversive learning. By exploiting our phosphoproteomics system for comprehensive PKC substrate screening, we found that acetylcholine activates Rho family GTPase Rac and its downstream effector PAK kinase. Also, we found that the activated Rac-PAK signaling by acetylcholine in striatopallidal medium spiny neuron contributes to aversive learning. This study would advance our understanding of the mode of action of Alzheimer's disease therapeutic drugs, shedding light to the molecular basis of Alzheimer's disease.

We have proposed that L-DOPA is a neurotransmitter. L-DOPA when exogenously microinjected into the nucleus tractus solitarii (NTS) induces depressor and bradycardic response. Electro-stimulation of aortic depressor nerves (ADN) induces L-DOPA release from the NTS, and induces depressor and bradycardic response. These our findings suggested that L-DOPA may play a neurotransmitter role in the primary baroreceptor afferents terminating in the NTS. To further determine the role of L-DOPA as a neurotransmitter in the ADN, we investigated whether depressor and bradycardic responses to L-DOPA was mimicked by stimulating ADN using optogenetic procedure in rats. We injected adeno-associated virus encoding ChR2-EYFP or EYFP into the ganglion of ADN. Four to five weeks after injection, ChR2-EYFP and EYFP signals were partially localized with tyrosine hydroxylase-positive neurons in the NTS of the fixed brain tissues. Photostimulation (473 nm, 40 mW, 20 Hz, 20s) from the surface of the NTS induces depressor and bradycardic response in the animals expressing ChR2-EYFP, but not EYFP in the ADN. The effects of photostimulation were attenuated by treatment with L-DOPA cyclohexylester (1 μg), an antagonist for L-DOPA, in the NTS, as were those of exogenously applied L-DOPA. These results suggested that L-DOPA is a neurotransmitter in the ADN terminating into the NTS.

Stress is one of the most frequently self-reported precipitants for seizure induction in epilepsy patients, but how stress triggers seizures remains unknown. In the medial prefrontal cortex (mPFC), stress has been known to enhance the release of noradrenaline (NA), which excites mPFC layer 5 (L5) pyramidal cells. Thus, we investigated the possible contribution of NA in the mPFC to stress-induced epileptic seizures. Intra-mPFC infusion of picrotoxin (0.1 nmol/side) and NA (10 nmol/side) induced seizures with shorter latency than that of picrotoxin alone in C57BL/6J mice. In vitro whole-cell patch-clamp recordings from mPFC L5 pyramidal cells revealed that, in the presence of picrotoxin (30 µM), bath-application of NA (10 µM) induced rhythmic and frequent epileptiform activities (EA) consisting of prolonged depolarization with burst firings in short latency, while picrotoxin alone induced sporadic and long-latency EA. The NA-induced EA were inhibited by terazosin (5 µM), but not atipamezole (3 µM) or timolol (10 µM), indicating the involvement of α1 adrenoceptors for the EA generation. These results suggest that NA released in the mPFC might contribute to the expression of stress-induced seizures in epilepsy patients.

Shati/Nat8l (Shati) is a novel N-acetyltransferase identified in the brain of mice treated with methamphetamine (METH). Recent studies showed that Shati overexpression in the nucleus accumbens (NAc) attenuates pharmacological response of methamphetamine (METH) via mGluR3. Meanwhile, it is reported that after METH self-administration and during reinstatement, dopamine (DA) and glutamate dysregulations were observed in medial PFC (mPFC) and NAc of rats. However, the regulatory mechanism of control over-reward system and the function of Shati in mPFC have not been clarified.

In this study, we injected AAV-Shati vector into the mPFC of mice. Interestingly, Shati injected mice attenuated METH-induced conditioned place preference (CPP) but not locomotor activity. Additionally, immunohistochemistry of mice injected with GFP in mPFC showed an mPFC-NAc top-down regulation. Finally, in vivo microdialysis experiments showed a decrease in DA baseline and METH-induced increased DA in the NAc. Moreover, a decrease in extracellular glutamate levels was also observed in the NAc.

These results suggest that Shati overexpression in mPFC attenuates METH-induced CPP by decreasing extracellular DA in the NAc.

Brain edema is a critical condition resulted from blood-brain barrier (BBB) disruption after traumatic brain injury (TBI). Several astrocyte-derived factors are involved in BBB function. We previously confirmed the involvements of endothelin ET_B receptor for BBB disruption. In this study, the effects of BQ788, an ET_B receptor antagonist on brain edema and astrocyte-derived factors were examined. As a model of TBI, fluid percussion injury (FPI) was performed on mouse cerebrum. BBB disruption and brain edema were evaluated by Evans blue extravasation and brain water content, respectively. Expressions of astrocyte-derived factors were confirmed by Real-time PCR and immunohistochemistry. To confirm therapeutic efficiencies, intraventricular (i.c.v., 15 nmol/day) or intravenous (i.v., 5 mg/kg/day) administration of BQ788 were performed from 2 to 5 days after FPI. BQ788 ameliorated FPI-induced BBB disruption and brain edema but not affected blood pressure. ET_B receptors were mainly distributed in astrocytes and BQ788 appropriately controlled expressions of several astrocyte-derived factors after FPI. Thus, blockade of ET_B receptor is expected to be a novel therapeutic strategy for brain edema.

Defective mitochondrial dynamics through aberrant interactions between mitochondria and actin cytoskeleton is increasingly recognized as a key determinant of cardiac fragility after myocardial infarction (MI). Dynamin-related protein 1 (Drp1), a mitochondrial fission–accelerating factor, is activated locally at the fission site through interactions with actin. Here, we report that the actin-binding protein filamin A acted as a guanine nucleotide exchange factor for Drp1 and mediated mitochondrial fission–associated myocardial senescence in mice after MI.Pharmacological perturbation of the Drp1–filamin A interaction by cilnidipine suppressed mitochondrial hyperfission–associated myocardial senescence and heart failure after MI. These data demonstrate that Drp1 association with filamin, and the actin cytoskeleton, contributes to cardiac fragility after MI and suggests a potential repurposing of cilnidipine.

Chronic volume overload to the heart has been suggested to generate arrhythmic substrates in the atria, leading to the onset of atrial fibrillation (AF). We investigated a role of TRPC channels in the pathophysiological process of atrial remodelings caused by aorto-venocaval shunt (AVS) in rats. More than 3 months after the surgery, the atrial weight of the AVS group (n = 10) was greater than that in the Sham group (n = 9), and fibrosis was observed in the atrial tissue. The P-wave duration, PR interval and QRS width of the ECG were significantly prolonged in the AVS group compared with those in the Sham group. The duration of AF induced by burst pacing in the AVS group was about 4 times longer than that in the Sham group. The mRNA levels of TRPC channel in the AVS group were significantly greater than those in the Sham group. Chronic administration of a TRPC 3 channel inhibitor pyrazole-3 to the AVS rats ameliorated the prolongation of the P-wave duration and QRS width caused by AVS surgery. Furthermore, the duration of AF was significantly shortened by the drug to the same level as the Sham group. These results suggest that TRPC3 channel is involved in the atrial remodeling caused by chronic volume overload.

Doxorubicin (Dox) is a highly effective anticancer agent, but eventually induces cardiotoxicity associated with increased production of reactive oxygen species (ROS). We previously reported that the formation of protein complex between transient receptor potential canonical 3 (TRPC3) and NADPH oxidase 2 (Nox2) mediates Dox-induced cardiac atrophy in mice. The aim of this study was to identify a drug already approved for clinical use that blocks Dox-induced cytotoxicity, and demonstrate whether inhibition of TRPC3-Nox2 complex by this drug attenuates Dox-induced systemic tissue wasting in mice. Drug screening was performed using Raw264.7 macrophage cell line. We investigated whether inhibition of TRPC3-Nox2 complex contributed to attenuation of Dox-induced cytotoxicity, by measuring Nox2 protein stability through interaction between TRPC3 and Nox2. We found that ibudilast, an anti-asthmatic drug, attenuated the cytotoxicity induced by Dox and cisplatin, by inhibiting TRPC3-Nox2 functional interaction without reducing TRPC3 channel activity. These results identify a common mechanism underlying induction of systemic tissue wasting by Dox and a drug that could be repurposed to reduce the risk of cardiotoxicity by anti-cancer drugs.

Anderson Fabry disease (AFD) is caused by mutations in the X-linked gene GLA, encoding lysosomal enzyme α-galactosidase A (α-GAL). The gene mutations reduce the enzymatic activity, resulting in significant lysosomal accumulation of enzyme substrates glycosphingolipids, mainly globotriaosylceramide (Gb3) and globotriaosylsphingosine (lyso-Gb3). However, how the accumulated glycosphingolipids cause disease phenotypes remain largely unknown. Here, we employed human induced pluripotent stem cells (iPSCs) modified with clustered regularly interspaced short palindromic repeats interference (CRISPRi) to model cardiac AFD in vitro. We found that clumps of iPS-derived cardiomyocytes (iPS-CMs) with CRISPRi-mediated GLA knockdown exhibited impaired beating, prolonged Ca²⁺ decay and relaxation. Mechanistically, α-GAL reduction increased unphosphorylated PLN by perturbing mTOR (specifically mTORC2, but not mTORC1)-AKT signaling. These results reveal how the pathogenic glycosphingolipids intersect with the mTORC2-AKT-PLN axis regulating relaxation and impair it, leading to the cardiac abnormalities. Our study reinforces the usefulness of the CRISPRi iPSCs in modeling monogenic diseases and delineating mechanisms of the diseases.

The lymphatic system is involved in pathogenesis of edema, inflammation and cancer metastasis. It has been reported that lymph capillary network regulates interstitial electrolyte and volume balance, which buffers blood pressure. Thus, we hypothesized that impaired lymphatics dysfunction leads to increasing blood pressure. In this study, we employed thoracic duct from 10-14-week-old Wister-Kyoto (WKY) and spontaneously hypertensive rats (SHR) and examined lymphatics contractility. Thoracic duct from SHR exhibited impaired acetylcholine (ACh)-induced endothelial-dependent relaxation compared to age-matched WKY (39.4±1.0% vs 84.8±2.2%, p<0.05). L-NAME, a NOS inhibitor blunted ACh-induced relaxation in WKY and SHR. Tempol, a superoxide dismutase mimetic significantly improved the impairment in SHR (p<0.05). Thoracic duct from SHR also displayed decreased SNP-induced relaxation (p<0.05). Despite the marked impairment in relaxation, contraction response to angiotensin II in SHR was similar to WKY. We conclude that lymphatic function was significantly blunted in hypertensive model at least SHR through superoxide. The impairment of the lymphatic relaxation may be involved in increased blood pressure in hypertension due to dysfunction of lymphatic buffering.

The ATP-binding cassette transporter G1 (ABCG1) is a cholesterol lipid efflux pump whose role in tumor growth has been largely unknown. Our transcriptomics revealed that ABCG1 was powerfully expressed in rapidly metastatic, aggregative colon cancer cells, in all the ABC transporter family members. Coincidently, genetic amplification of ABCG1 is found in 10% to 35% of clinical samples of metastatic cancer cases. Expression of ABCG1 was further elevated in three-dimensional tumoroids (tumor organoids) within stemness-enhancing tumor milieu, whereas depletion of ABCG1 lowered cellular aggregation and tumoroid growth in vitro as well as hypoxia-inducible factor 1α in cancer cells around the central necrotic areas in tumors in vivo. Notably, depletion of ABCG1 triggered the intracellular accumulation of extracellular vesicles (EVs) and regression of tumoroids. Collectively, these data suggest that ABCG1 plays a crucial role in tumorigenesis in metastatic cancer and that depletion of ABCG1 triggers tumor regression with the accumulation of EVs, their derivatives and cargos, implicating a novel ABCG1-targeting therapeutic strategy by which redundant and toxic substances may be accumulated in tumors leading to their regression.

Angiogenesis, a process of vascular growth from preexisting vessels, is involved in pathological processes including tumor growth and metastasis. L-type amino acid transporter 1 (LAT1) is highly expressed in a wide range of cancers and contributes to supplying amino acids. LAT1 has, thus, been proposed as a potential target for cancer therapy. Recently, we found that LAT1 shows high expression not only in tumor cells but also in endothelial cells of pancreatic cancer tissues. In contrast, the expression of LAT1 in endothelial cells of normal tissues is limited. In this study, we examined whether LAT1 is involved in tumor angiogenesis or not. LAT1 inhibitors reduced the number of blood vessel sprouting in ex vivo aortic ring assay. Suppression of angiogenesis by LAT1 inhibitors was further confirmed in in vivo matrigel plug assay and xenograft tumor model. Moreover, contribution of LAT1 in angiogenesis was verified using human umbilical vein endothelial cells in in vitro angiogenesis assays. LAT1 inhibitors are supposed to exert an anti-angiogenic effect through the inhibition of LAT1 in the endothelial cells of tumors, which could, together with the suppression of amino acid uptake of cancer cells, contribute to the anti-tumor effect in vivo.

Tumor microenvironment (TME) is hypoxic and low pH. TME confer not only resistance of anti-cancer drug therapy on tumor cells, but also impair the activity of immune cells. Under TME condition, tumor-infiltrated macrophages (Mfs) change phenotype from anti-tumor (M1) phenotype to tumor progressive (M2) phenotype. Previously we reported that prolyl hydroxylase (PHD) inhibitor, which activate hypoxia-induced factors (HIFs), improve TME. In this study, we examined whether the improvement of TME by PHD-inhibitor activate tumor-infiltrated Mfs. Lewis lung carcinoma cells were subcutaneously transplanted into right frank of mice which aged at 8-12 weeks. Mice were treated with PHD inhibitors intraperitoneally at day 10 after tumor transplantation. Then tumor tissues were collected at day 16 and analyzed immune cells by immunofluorescence staining and flowcytometry. We performed phagocytosis assay using sorted Mfs from tumor tissue and bone derived Mfs. Furthermore, we injected sorted Mfs into the tumor and evaluated tumor growth. PHD-inhibitor inhibited tumor growth. We concluded that PHD inhibitor directly activated Mfs and TME improvement supported activity of Mfs.

G protein-coupled receptor (GPCR) kinase 2 (GRK2) has emerged as an integrating node in many signaling pathways besides its GPCR signaling role. We investigated how GRK2 could contribute to Toll-like receptor (TLR4) signaling for iNOS expression in microglia. We found in microglial cells LPS-induced iNOS expression requires both STAT1 and STAT3 activation. GRK2 knockdown by siRNA transfection suppressed phosphorylation and nuclear translocation of STAT1/3. Interferon-β (IFN-β) produced from TLR4 signaling induce phosphorylation of STAT1/3, and its expression is regulated by interferon regulatory factors (IRF) at transcription level. We found that GRK2 knockdown suppressed LPS-induced IFN-β production and mRNA expression. LPS stimulation induce IRF1 expression and nuclear translocation, and GRK2 knockdown suppressed IRF1 nuclear translocation without affecting expression level. These results suggest that GRK2 positively regulates IRF1 activation to induce IFN-β expression, STAT1/3 activation, and subsequently iNOS expression. Furthermore, GRK2 knockdown suppressed exogenous IFN-β-induced STAT1/3 activation. In conclusion, GRK2 upregulates TLR4-induced iNOS expression by mediating IRF1 and STAT1/3 activation.

Genome is spatially organized in the nucleus. The chromatinized DNA segregates into an active "A" compartment, which contains gene-dense chromatin bearing active epigenetic marks, and an inactive "B" compartment, which contains gene-poor chromatin bearing repressive epigenetic marks. Compartments represent the long-distance interaction patterns including topologically associating domains (TADs) and lamina-associated domains (LADs). Influenza virus replicates its genome in the nucleus and viral proteins have shown to interact with multiple components of the host epigenetic machinery to promote viral replication and limit host immune responses. However, it is unknown how influenza virus infection can affect the spatial configuration of chromatin and function of the host genome in the nucleus. In the present study, using genome-wide chromosome conformation capture (Hi-C) technique we showed the dynamic changes in TADs and A/B formations during the course of influenza virus infection in mouse embryonic fibroblasts. In addition to nuclear lamina disruption, DamID-seq demonstrated the changes in LADs formation following virus infection. Thus, our data suggest dynamic changes in host genome 3D structures to virus infection, which could be a novel therapeutic target for influenza virus infection.

Ca²⁺-sensing receptor (CaSR) is a seven-transmembrane G protein-coupled receptor (GPCR), and is activated by an increase in extracellular Ca²⁺ concentration. In vascular endothelial cells, stimulation of CaSR induces nitric oxide (NO) release via activation of endothelial nitric oxide synthase (eNOS) and membrane hyperpolarization via activation of intermediate Ca²⁺-activated K⁺ channels, contributing to vasodilation. In the present study, we have pharmacologically characterized eNOS activation in response to stimulation of CaSR in human endothelial EA.hy926 cells. In 2 mM Ca²⁺-containing Krebs-HEPES solution, the phosphorylation level of eNOS at serine 1177 was markedly reduced by NPS 2143 (a CaSR antagonist) and YM-254890 (a G_q/11 protein inhibitor). In organ bath study with endothelium-removed ring preparations of rat thoracic aorta, addition of EA.hy926 cell suspension produced relaxation of the rings precontracted with phenylephrine. The endothelium-dependent relaxant response was inhibited by pretreatment of EA.hy926 cells with NPS 2143, YM-254890, and L-NAME (an eNOS inhibitor). These results suggest that stimulation of CaSR expressed in endothelial cells with extracellular Ca²⁺ induces NO-mediated vasorelaxation via G_q/11-protein-dependent activation of eNOS.

Endothelial cell dysfunction underlies the development of vascular inflammatory diseases. The cellular stiffness of endothelial cells has been shown to increase during chronic inflammation. Abnormalities of endothelial gap junctions facilitate endothelial dysfunction and participate in the progression of cardiovascular diseases; however, the impact of gap junction on endothelial cellular stiffness remains poorly understood. 

To evaluate the cellular stiffness, we have measured the force curve of live human umbilical vein endothelial cells (HUVECs) by using atomic force microscopy. We have shown that stimulation of HUVECs with tumor necrosis factor-α (TNF-α) increased the endothelial cellular stiffness. We have also shown that the blockade of gap junctions not only increases endothelial cellular stiffness, but also enhances focal adhesion formation and cytoskeletal rearrangement. Inhibition of gap junctions appeared to prolong the effects of TNF-α, increasing endothelial stiffness. 

Aberrant regulation of gap junctions may be involved in the vascular stiffening commonly observed in vascular inflammatory diseases. Our study provides a new insight into the potential pathogenic role of endothelial cellular stiffening driven by vascular inflammation.

Diabetes induces proangiogenic response which is characterized by fragile blood vessels. Advanced glycation end products (AGEs) accumulating under hyperglycemic conditions are acknowledged to play a causative role in the vascular complications of diabetes including retinopathy associated with excessive angiogenesis. However, the detailed mechanism of AGEs-mediated excessive angiogenesis is poorly understood. Therefore, we elucidate one part of this mechanism. Matrigel tube formation assay was performed using the mouse endothelial cell line, b.End5 cells. The areas and total lengths of the tubes were calculated as the degree of tube formation. Uptake of AGEs by b.End5 cells were measured using flow cytometry. pNFκB/NFκB ratio were analyzed by western blot. AGE2 and AGE3 concentration-dependently increased the area and length of tubular structures. Uptake of AGE2 and AGE3 by b.End5 cells concentration-dependently was increased. AGE2 and AGE3 activated NFκB. Induction of tube formation by AGE2/3 and AGE2/3 uptake by b.End5 cells were significantly suppressed by pinocytosis inhibitor, EIPA, or NFκB inhibitor, PDTC. Our results indicate that AGEs uptake by b.End5 cells may be mediated by pinocytosis. Moreover, pinocytosis of AGEs may induce phosphorylation of NFκB in endothelial cells, and then promote angiogenesis.

Microsomal prostaglandin E synthase-1 (mPGES-1) /Prostaglandin E2 (PGE₂) induces angiogenesis.Immune cells, especially regulatory T cells (Treg) related to cancer growth and angiogenesis. Based on these previous reports, we hypothesized mPGES-1/PGE₂-EP signaling contribute to recovery from ischemic condition by accumulation of Treg.

Compared to wild type mice (WT), the blood recovery was significantly suppressed in mPGES-1 deficient mice (mPGES-1KO). The number of FOXP3⁺cells in the ischemic muscle was decreased in mPGES-1KO compared to WT. Expression of TGF-beta was suppressed in mPGES-1KO. Those accumulated FOXP3⁺cells and blood recovery was significantly suppressed by injecting folate receptor 4 (FR4) antibody in WT (P<0.05) but not in mPGES-1KO. Compared to other EP receptors, the expression of EP4 in the ischemic muscle was significantly enhanced . The blood recovery was significantly suppressed in EP4 receptor deficient mice (EP4KO) compared to WT (P<0.05). Furthermore, expression of mRNA level of FOXP3 and TGF-beta were significantly suppressed in EP4KO. Moreover, the numbers of FOXP3+ cells were diminished in EP4KO compared to WT. These results suggested that mPGES-1/PGE₂induces blood flow recovery from ischemia via EP4 by accumulating Tregs.

Due to atherosclerosis is asymptomatic nature, it is difficult to investigate the progression of this disease in humans. Animals, especially mice with select genetic alterations, are very useful to investigate this disease. Mice with a double deficiency of LDLr and Apobec1 (Ldlr^-/-/Apobec1^-/-) show high levels of plasma total cholesterol on a normal chow diet, most of which is distributed in the LDL fraction. Consequently, spontaneous atherosclerotic plaques form in the aorta, a situation that is similar to human familial hypercholesterolemia.

We have revealed the important role of thecoagulation and fibrinolytic system in atherosclerosis. In this study, we examined the role of plasminogen in atherosclerosis with Ldlr^-/-/Apobec1^-/-mice.

We established Ldlr^-/-/Apobec1^-/-/Plasminogen^-/-(Ldlr^-/-/Apobec1^-/-/Plg^-/-) triple deficient mice from Ldlr^-/-/Apobec1^-/-mice and Plg^-/-mice. We have found that total cholesterol levels were significantly higher in Ldlr^-/-/Apobec1^-/-/Plg^-/-mice than in Ldlr^-/-/Apobec1^-/-mice. Almost all of the cholesterol accumulated in the LDL fraction, and the HDL cholesterol level was not different between both groups. However, Ldlr^-/-/Apobec1^-/-/Plg^-/-mice showed much smaller plaques in the aortic sinus. Furthermore, the migration of macrophages was suppressed by a plasminogen deficiency. Plasmin, the activator of plasminogen, promoted OxLDL uptake of macrophages. On the other hands, plasminogen didn't affect the expression of various scavenger receptors in macrophage. These results suggest that theactivation of plasminogen and the interaction plasminogen and OxLDL promote atherosclerosis in type lla familial hypercholesterolemia.

Cystic fibrosis (CF) is typically characterized by infection-associated airway epithelial inflammation. Here, we showed that cell surface expression of SIGIRR/TIR8, an immunoglobulin-like membrane protein that is essential for negative regulation of toll-like receptors (TLRs)-associated inflammatory signals, is specifically and remarkably decreased in CF and CF-like airway epithelial cells. These CF-associated cells specifically and highly expressed a unique, alternative splice isoform of SIGIRR that lacks exon 8 (Δ8-SIGIRR),

which contributes to the suppression of functional SIGIRR plasma membrane expression. Consistently, a SIGIRR ligand IL-37b failed to suppress inflammatory signals induced by poly(I:C), a synthetic analog of viral RNA. Finally, poly(I:C)-dependent anti-inflammatory IL-37b secretion was also significantly decreased in CF cells, suggesting the dysregulation of anti-inflammatory receptor SIGIRR as well as its ligand IL-37b in CF cells. Thus, our studies demonstrate that poly(I:C)-dependent anti-inflammatory feedback loop, or IL-37b-SIGIRR negative feedback signal, is defective in CF airway epithelial cells due to unique splicing switch of SIGIRR gene.

Idiopathic pulmonary fibrosis (IPF) is a chronic and progressive lung disease with a poor prognosis. Thus, to discover drugs providing new therapeutic options for patients with IPF is necessary. Myofibroblasts (MyoFs) that produce abundant extracellular matrix play key roles in the development of IPF. Although the pathogenic MyoFs mainly differentiated from resident fibroblasts have been considered as the irreversible phenotype, it has been recently reported that some agents dedifferentiating MyoFs into fibroblasts can attenuate bleomycin-induced lung fibrosis in mice. This finding tempts us to consider induction of MyoF dedifferentiation as a new beneficial strategy for patients with IPF. However, the detailed mechanisms of MyoF dedifferentiation are still unknown.

We have already established several strains of primary cultured MyoF-like cells (MyoLCs) from the fibrotic lungs of patients who underwent lung transplantation as a result of severe fibrosis. Employing the MyoLCs (S100A4^-α-SMA^highED-A-fibronectin⁺: > 90%) as an in vitro screening system, we found that JQ1, a bromodomain inhibitor could induce MyoF dedifferentiation associated with the downregulation in expression of α-SMA and ED-A fibronectin. Then, we comprehensively investigated the change in expression of microRNAs in JQ1-treted MyoLCs. Based on these results, we will discuss the molecular mechanisms of MyoF dedifferentiation.

Calcitonin gene-related peptide (CGRP) regulates inflammation through receptor activity-modifying protein 1 (RAMP1), a subunit of CGRP receptor complex in immune cells. In this study, we examined the role of RAMP1 in immune-mediated liver injury. RAMP1-knockout (RAMP1^-/-) mice or their wild-type counterparts (WT) were treated with Concanavalin A (Con A). Compared with WT, RAMP1^-/- mice exhibited higher levels of ALT, necrotic area, and the mRNA for pro-inflammatory cytokines including TNF and IFN. The numbers of macrophages and T cells in RAMP1^-/- mice were greater than those in WT mice. Deletion of hepatic macrophages with clodronate liposomes attenuated ALT and necrotic area in both genotypes as compared with vehicle, which was associated with reduction in TNF and IFN. By contrast, splenectomy and deletion of T cells with anti-CD4 antibody partly attenuated Con A hepatitis. Adoptive transfer of splenic T cells from RAMP1-deficient mice led to a modest increase in liver injury elicited by Con A. Co-culture of hepatic macrophages with splenic T cells led to increased cytokine expression by both cells in a RAMP1-dependent manner. Thus, immune-mediated hepatitis develops via crosstalk between immune cells. RAMP1 signaling in hepatic macrophages plays a critical role in immune-mediated hepatitis.

Cancer patients often develop anorexia during the course of cancer chemotherapy. It is known that daytime sleepiness is also the major complaints made by cancer patients, and this sleep problem can worsen anorexia. Previous studies reported the histamine H₃ receptor as a target for treating sleep disorders, and selective H₃ receptor inverse agonists are effective at reducing daytime sleepiness. In this study, we investigated the involvement of the H₃ receptor in the development of chemotherapy-induced anorexia in mice. Cisplatin (7.5 mg/kg, i.p.) induced anorexia within 24 hours of its administration and it continued for 3 days, and daily administration of an H₃ receptor inverse agonist (ciproxifan, 1 mg/kg, s.c.) significantly inhibited the development of anorexia. Daily administration of an orexin 2 receptor agonist (YNT-185, 20 mg/kg, s.c.), which was reported to induce wakefulness in mice and to activate the histaminergic system, also inhibited the cisplatin-induced anorexia, and its inhibitory effects were antagonized by daily administration of an H₃ receptor silent antagonist (VUF5681, 5 mg/kg, s.c.). These results suggest that sleep problem may contribute to the development of cisplatin-induced anorexia in mice, and H₃ receptor inverse agonists have the potential to be candidates used as its treatment.

Properties of spontaneous electrical activity in the mouse proximal colon were studied using intracellular recording. Spike complexes with a frequency of 1 ～ 3 cycles/min occurred in the longitudinal muscles. Each spike complex consisted of a burst of action potentials (APs) 15 ～ 35 mV in amplitude and 10 ～ 40 APs per complex. The resting membrane potentials were -40 ～ - 50 mV. Spike complexes were abolished by verapamil, a Ca²⁺ blocker, pinacidil, a K_ATP channel opener, NPPB, a Ca²⁺ - activated Cl^- channel (CACC) blocker, and bumetanide, a Na⁺ - K⁺ - 2Cl^- co-transporter (NKCC1) inhibitor. Spike complexes and spontaneous transient depolarizations (STDs) were recorded from myenteric interstitial cells of Cajal (ICC-MY). Spike complexes in ICC-MY were also abolished by verapamil, pinacidil, NPPB and bumetanide. The amplitude of STDs were inhibited by verapamil and NPPB, but increased by pinacidil. These results suggest that the activation of CACC is associated with the generation of spontaneous electrical activity in the mouse proximal colon. Since CACC and NKCC1 are expressed only in ICC-MY, spike complexes seem to be generated in ICC-MY and transferred to nearby smooth muscle layers electrotonically.

SGLT2 inhibitors lower blood glucose levels by preventing renal glucose reuptake, but they often cause hyperglucagonemia. Recent studies suggested its expression and potential role as a glucagon suppressor in pancreatic alpha cells, though it has been under debate. Thus, we conducted functional analyses of SGLT2 in a typical model of pancreatic alpha cell, α-TC cells to unveil roles of SGLT2 in the glucagon secretion. Glucagon secretion as well as intracellular ATP level decreased in response to glucose deprivation. SGLT2 inhibitors reduced glucose uptake, but glucagon secretion nor ATP level was affected. An inhibitor of KATP channel increased glucagon secretion without changing ATP level. Therefore, glucose starvation should not facilitate but mitigate glucagon secretion in α-TC cells possibly by raising AMP/ATP ratio which mitigates membrane potential through KATP channel. We also found SGLT2-mediated glucose uptake in α-TC cells. Nevertheless, the glucose influx is supposed to be too small to take effects on ATP level, and SGLT2 inhibitors should not directly alter glucagon secretion. Glucose starvation-induced glucagon secretion may require interaction among different types of the cells in islets.

Overactivation of poly (ADP-ribose) polymerase-1 (PARP-1) has been implicated in the pathogenesis of oxidative stress-related diseases, including diabetes and its complications. Obesity is linked with type 2 diabetes. We previously reported that thiamine supplementation prevents obesity and diabetes-related liver disease. In this study, we focused on hepatic ADP-ribosylation, which reflects the activation of PARP-1. Obese diabetic Otsuka Long-Evans Tokushima fatty (OLETF) rats were randomly divided into two groups: a thiamine-supplemented group and an unsupplemented control group. ADP-ribosylated protein expression in the liver was determined by western blotting. Obese diabetic OLETF rats showed increased ADP-ribosylated protein expression in the liver. Hepatic ADP-ribosylated protein expression in thiamine-supplemented OLETF rats was lower than that in the unsupplemented control OLETF rats. These results suggest that thiamine supplementation modulates oxidative stress by inhibiting hepatic ADP-ribosylation in OLETF rats. The beneficial effect of high-dose thiamine on oxidative stress-related diseases may also be attributable to its inhibitory effect on PARP-1activation in addition to its role as a coenzyme.

In our previous study, we extracted many genes commonly up-regulated by PPARα agonists (fibrates) in the rat liver using the transcriptome database created by The Toxicogenomics Project (TG-GATEs). Of these, nicotinic receptor α2 subunit (Chrna2) and acetylcholine (Ach) esterase anchoring protein (Colq) were markedly up-regulated by fibrates without any changes in other Ach receptors. Their induction was confirmed by quantitative PCR in the rats received fenofibrate for 3 days. The expression of Chrna2 was largely increased with repeated administration but not observed in the primary cultured hepatocytes. In the meantime, it was reported that Chrna2 was up-regulated in PPARγ-activated beige adipocytes and Ach released from immune cells stimulated the receptor to induce thermogenic protein, UCP1（Nature Med. 24 814, 2018).  We then checked UCPs and found that UCP3, not UCP1, was markedly up-regulated by fibrates. The present data suggest that hepatocytes have a specific energy consuming system, PPARα>Chrna2>UCP3, similar to but different from that in adipocytes.

Lipid droplet (LD) is surrounded by phospholipid monolayer mainly composed of phosphatidylcholine (PC). Steroidogenic acute regulatory protein (StAR)-related lipid transfer domain containing 10 (STARD10) has been shown to transfer PC between membranes in vitro. Lysophosphatidylcholine acyltransferase1 (LPCAT1) catalyzes the conversion of lysoPC to PC. The purpose of this study was to elucidate the role of STARD10 and LPCAT1 in LD formation. We hypothesized that the LD size depends on the surface-to-volume ratio. STARD10 and LPCAT1 were partly co-localized at the surface of LD in mouse hepatoma cells (Hepa1-6). The number of small LDs was increased by LPCAT1 overexpression, while the number of large LDs was increased by the overexpression of both STARD10 and LPCAT1. The percentage of small LDs was significantly higher in Stard10 knockout Hepa1-6 cells than that of normal Hepa1-6 cells. In the liver of Stard10 knockout mice, the total LD area was smaller and the sphericity of LD was lower than those of wild type mice, indicating that surface-to-volume ratio was higher. These results indicate that STARD10 and LPCAT1 are involved in LD formation by regulating surface-to-volume ratio through its activity of PC transfer and synthesis.

[Introduction] With the increase of obese and diabetic patients, anti-obesity drugs which decrease fat content without muscle atrophy are expected. In obese or diabetic patients, serum copper concentration was reported to increase compared with that in healthy subjects. However, it is still unknown whether the lowering of serum copper levels has anti-obese effects. Therefore, we investigated the effects of copper chelator, cuprizone on high fat diet (HFD)-induced obesity in mice model.

[Method] We administered cuprizone (0.2%w) mixed in food pellet. Mice fed (1) normal chow (NC), (2) NC with cuprizone, (3) HFD or (4) HFD with cuprizone for 4 weeks, and then metabolic parameters were obtained.

[Results] Serum copper level was decreased in both cuprizone groups. Cuprizone significantly decreased the body weight extensively in HFD, but slightly in NC fed mice, without changes of food intake. Interestingly, cuprizone specifically decreased 60% of epididymal and inguinal fat weights, but not liver and muscle (soleus and gastrocnemius) weights, only in HFD. Furthermore, HFD-induced glucose intolerance (ipGTT) and insulin resistance (ITT) were significantly improved by cuprizone

[Conclusion] Cuprizone inhibited HFD-induced adiposity via the decreased amount of fat depot.

Although increased production of prostaglandin E₂ (PGE₂) has been implicated in tissue damage in several pathological settings, the role of microsomal prostaglandin E synthase-1 (mPGES-1), an inducible terminal enzyme for PGE₂ synthesis, in dopaminergic neurodegeneration remains unclear. Here we show that mPGES-1 is up-regulated in the dopaminergic neurons of the substantia nigra of postmortem brain tissue from PD patients and in 6-hydroxydopamine (6-OHDA)-induced PD mice. The expression of mPGES-1 was also up-regulated in cultured dopaminergic neurons stimulated with 6-OHDA. The genetic deletion of mPGES-1 not only abolished 6-OHDA-induced PGE₂ production but also attenuated 6-OHDA-induced dopaminergic neurodegeneration both in vitro and in vivo, while it did not affect the productions of PGI₂, PGD₂ and TXA_2. Nigrostriatal projections, striatal dopamine content, and neurological functions were significantly impaired by 6-OHDA administration in wild-type mice, but not in mPGES-1 knockout mice. These results suggest that induction of mPGES-1 in dopaminergic neurons enhances 6-OHDA-induced dopaminergic neurodegeneration through excessive PGE₂ production. Thus, mPGES-1 may be a valuable therapeutic target for treatment of PD

Parkinson's Disease (PD) is one of the most common neurodegenerative diseases, resulting from degeneration of dopaminergic neurons in substantia nigra. Although more than twenty causative genes have been identified to date, the majority is sporadic and the detailed pathological mechanism remains unclear. We identified a novel PD-associated gene, Midnolin (MIDN) by Yamagata cohort study. The copy number of MIDN was reduced in 10.5% of patients with sPD, whereas no copy number variation was found in healthy people. Therefore, we examined the pathophysiological roles of MIDN in this study. NGF caused gene expression of MIDN in a time- and concentration-dependent manner in PC12 cells. NGF-induced neurite outgrowth was completely inhibited in PC12 cells where MIDN gene was knocked out by genome-editing. Furthermore, we found that mRNA and protein expression of parkin E3 ubiquitin ligase was inhibited by MIDN knockout or siRNA targeting MIDN in PC12 cells and SH-SY5Y cells. These results suggest that the loss of MIDN gene causes inhibition of neurite outgrowth and parkin expression, which may result in the onset of PD.

To realize cell transplantation therapy for Parkinson's disease (PD), the grafted neurons should be integrated into the host neuronal circuit in order to restore the lost neuronal function. Here, using wheat germ agglutinin-based trans-synaptic tracing, we show that integrin α5 is selectively expressed in striatal neurons that are innervated by midbrain dopaminergic (DA) neurons. Additionally, we found that integrin α5β1 was activated by the administration of estradiol-2-benzoate (E2B) in striatal neurons of adult female rats. Importantly, we observed that the systemic administration of E2B into hemi-parkinsonian rat models facilitates the functional integration of grafted DA neurons derived from human induced pluripotent stem cells into the host striatal neuronal circuit via the activation of integrin α5β1. Finally, methamphetamine-induced abnormal rotation was recovered earlier in E2B-administrated rats than in rats that received other regimens. Our results suggest that the simultaneous administration of E2B with stem cell-derived DA progenitors can enhance the efficacy of cell transplantation therapy for PD.

A GGGGCC repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce dipeptide repeat proteins. Although it has been reported that dipeptide repeat proteins cause neurotoxicity, the underlying mechanism has not been fully elucidated. In this study, we show that the expression of proline-arginine repeat protein (poly-PR) reduces levels of ribosomal RNA and causes neurotoxicity. The poly-PR-induced neurotoxicity is restored by the acceleration of ribosomal RNA synthesis. This result suggests that the poly-PR-induced inhibition of ribosome biogenesis contributes to the poly-PR-induced neurotoxicity. Furthermore, we show that poly-PR interacts with multiple DEAD-box RNA helicases and inhibits the function of at least one of them, and that the reduction in the levels of some RNA helicases results in both the decrease in ribosomal RNA levels and the increase in neuronal cell death. Altogether, these results suggest that poly-PR causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.

Hydrogen sulfide (H₂S) and polysulfides (H₂S_n) are signalling molecules that mediate various physiological responses including cytoprotection. Their oxidized metabolite sulfite (SO₃^2-) is found in blood and tissues. However, its physiological role remains unclear. In this study, we investigated the cytoprotective effect of sulfite on neurons exposed to oxidative stress caused by high concentrations of the neurotransmitter glutamate, known as oxytosis. Free sulfite, present at approximately 2 μM in the rat brain, converts cystine to cysteine more efficiently than H₂S and H₂S_n and facilitates transport of cysteine into cells. Physiological concentrations of sulfite protected neurons from oxytosis and were accompanied by increased intracellular concentrations of cysteine and GSH probably due to converting extracellular cystine to cysteine, more efficiently than H₂S and H₂S_n. In contrast, thiosulfate only slightly protected neurons from oxytosis. Our present data have shown sulfite to be a novel cytoprotective molecule against oxytosis, through maintaining cysteine levels in the extracellular milieu, leading to increased intracellular cysteine and GSH. Our results provide a new insight into the therapeutic application of sulfite to neuronal diseases caused by oxidative stress

α-Melanocyte-stimulating hormone (α-MSH) is an endogenous peptide hormone that is involved in cutaneous pigmentation. Recent our study has demonstrated that α-MSH elicits scratching, an itch-related response, in mice. In this study, we investigated whether α-MSH was involved in spontaneous scratching in mice with atopy-like dermatitis (AD-mice). α-MSH and the prohormone convertase 2, which is the key processing enzyme for the production of α-MSH, were distributed mainly in keratinocytes in AD-mice. In primary cultures of mouse keratinocytes and dorsal root ganglion (DRG) neurons, α-MSH receptors (MC1R and MC5R) mRNAs were expressed. MC1R antagonist agouti-signaling protein inhibited spontaneous scratching in mice with atopy-like dermatitis. In mice, α-MSH elicited itch-associated responses, which were inhibited by TP thromboxane (TX) receptor antagonist. In mouse keratinocytes, α-MSH increased the production of TXA₂, which was decreased in mouse keratinocytes treated with siRNA for MC1R and/or MC5R. α-MSH increased intracellular Ca²⁺ ion concentration in DRG neurons and keratinocytes. These results suggested that α-MSH-TXA₂ system is involved in spontaneous scratching in AD-mice.