We have previously reported that nicotine (Nic) facilitates object recognition memory (ORM) encoding via activation of excitatory neurons in the medial prefrontal cortex (mPFC) in mice. In this study, we investigated whether Nic also facilitates ORM retrieval using the novel object recognition test (NOR). Male C57BL/6J mice (7 – 12 weeks) received Nic before the test session, which was performed 24 hours after the training session of the NOR. Systemic administration (0.1 mg/kg; s.c.), but not intra-mPFC infusion (0.3 μg/side), of Nic enhanced ORM, suggesting that Nic facilitates ORM retrieval by acting on brain regions other than the mPFC. However, suppression of mPFC neuronal activity with inhibitory DREADD hM4Di, which was specifically expressed in excitatory neurons using an AAV vector, significantly inhibited the systemic Nic-induced facilitation of ORM retrieval. Moreover, activation of mPFC excitatory neurons with excitatory DREADD hM3Dq significantly facilitated retrieval of ORM. These data suggest that Nic facilitates ORM retrieval through the indirect activation of mPFC excitatory neurons.

Cannabinoid receptor type 2 (CB2) is one of the major receptors for cannabis, which is expressed all over the body especially on the immune-related cells. It is considered that CB2 can work as a regulator in the immune system, particularly to suppress inflammatory responses in macrophages and microglias when activated. However no other immune cells have been studied whether and how they get modulated by CB2 activity. The objective of this experiment is to search for the other immune cell that can be affected by CB2 activity, and reveal the detailed mechanism of CB2-involved immune regulation.

To characterize the CB2-regulated cells under the inflammatory state, we injected LPS (1 mg/kg, i.p.) to CB2 deficient mice (CB2-KO) and wild-type (WT) controls. Spleen has been harvested from these mice 2 hours after the LPS administration, and immunophenotyping by flow cytometry has been conducted. In this experiment, we found the particular increase of the type 2 classical dendritic cell (cDC2) population in LPS-treated CB2-KO. As past studies reported that cDC2 recruits helper T cells when the body is exposed to an allergen, we further induced allergic rhinitis by OVA sensitization to investigate in-vivo effect of cDC2 increase in CB2-KO and WT animals. As expected, CB2 deficiency resulted as the significant exacerbation of allergic symptoms compared to WT mice. These results suggest that CB2 activity may suppress the allergic response by the reduction of cDC2 recruitment. Pharmacological effect of CB2 agonists to these symptoms will be studied as the future experiment.

In this study, we examined the selectivity, electrophysiological properties and analgesic activity of KTtp38, a novel inhibitor of T-type Ca²⁺ (Ca_v3) channels, developed by structural modification of pimozide, a typical antipsychotic agent. The IC₅₀ value (μM) of KTtp38 was 0.0934 and 1.109 for inhibiting Ca_v3.2-dependent currents in response to a test pulse of -20 mV from holding potentials (HPs) of -80 and -110 mV, respectively, indicating a state dependency. The IC₅₀ of KTtp38 for inhibiting Ca_v3.1-depedent currents caused by the test pulse from HP of -80 mV was 0.217 μM. Pimozide, but not KTtp38, at 1 μM completely inhibited the specific bindings of [³H]-spiperone to D₂ and D₃ receptors in rat striatal membrane fractions. In isolated rat jugular vein rings, the 5-HT₂ receptor-mediated contraction was inhibited by pimozide, but not KTtp38, at 10 μM. In mice, i.p. administration of pimozide, but not KTtp38, caused catalepsy. KTtp38 abolished somatic and visceral pain caused by an H₂S donor, known to enhance Ca_v3.2 activity, in mice. KTtp-38 also reversed oxaliplatin-induced peripheral neuropathy in wild-type, but not Ca_v3.2-null, mice. The T_1/2 (h) of KTtp38 and pimozide in the blood was 2.42 and 2.47, respectively. Collectively, KTtp-38 is considered a state-dependent, selective Ca_v3 inhibitor and useful as an analgesic.

Pulmonary arterial hypertension (PAH) is a progressive and fatal disease of the cardiovascular system. PAH is characterized by thickening of the pulmonary artery wall (remodeling) and often causes inflammation around the pulmonary artery. It has been reported that corosolic acid (CRA), is a pentacyclic triterpene acid contained in banaba leaves, has anti-inflammatory, anti-diabetic, and anti-cancer effects. In the present study, the effects of CRA on the pathogenesis of PAH were examined using monocrotaline (MCT)-induced pulmonary hypertensive (PH) rats. Male SD rats (4 weeks-old) were injected subcutaneously with vehicle (saline; control) or MCT (60 mg/kg). CRA (1 mg/kg) was administered intraperitoneally daily from 1 week after MCT injection. At 3 weeks after MCT injection, the effects of CRA on the in vivo parameters of PAH pathogenesis were analyzed. Hematoxylin and eosin (H&E) staining revealed that CRA clearly improved PAH remodeling in MCT-induced PH rats. The treatment with CRA also reduced the Fulton ratio (an index of right ventricular hypertrophy) in MCT-induced PH rats. Furthermore, CRA significantly lowered right ventricular systolic pressure (RVSP) in MCT-induced PH rats. In contrast, CRA did not affect these parameters in control rats. Taken together, CRA may be useful as a novel therapeutic candidate for PAH.

[Background] Honokiol is a small-molecule polyphenol isolated from the genus Magnolia and known as an activator of sirtuin 3, a mitochondrial deacetylase.  We examined whether honokiol attenuates mitochondrial injury, leading to the attenuation of cell death in hypoxia-reoxygenation (H/R) injury in cardiomyocytes. [Methods and Results] Neonatal rat cultured cardiomyocytes were subjected to 5 hours of hypoxia followed by 30 minutes of reoxygenation in the presence or absence of honokiol (30 μmol/L). Lethal myocyte injury was assessed by LDH activity in culture medium and myocyte apoptosis was examined by nuclear staining with DAPI and caspase 3 activity. H/R significantly increased LDH activity and apoptotic myocytes, and treatment with honokiol significantly attenuated these indices of myocyte death.  In mitochondrial apoptotic pathway, reduction of mitochondrial membrane potential plays critical roles, and ATP is mainly produced by mitochondria.  After H/R mitochondria lost their membrane potential, detected by TMRM fluorescence, leading to reduction of ATP content in myocytes, and honokiol recovered them. After H/R protein expression of sirtuin 3 was significantly restored by honokiol.  Sirtuin 3 is known to deacetylate Mn-SOD. After H/R honokiol decreased acetylation levels of Mn-SOD and tended to attenuate mitochondrial hydrogen peroxide production.  [Conclusion] These results indicate that Honokiol protects mitochondria via enhancement of sirtuin 3, leading to attenuation of H/R-induced myocyte lethal injury.

Aortic dissection is highly lethal, and the risk factors such as hypertension, aging, and atherosclerosis are thought to contribute to its onset. Recently, there has been increasing reports that vascular endothelial growth factor (VEGF) inhibitors can induce the aortic dissection as an adverse event. However, the association between VEGF inhibitors and aortic dissection has been unclear. Therefore, we investigated if VEGF inhibitor increases the onset of aortic dissection using acute aortic dissection model mice (AAD mice).

Sunitinib (100 mg/kg/day) was administered orally for 28days to AAD mice induced by nitric oxide inhibitor, angiotensin II, and lysyl oxidase inhibitor. Blood pressure was measured every week. After 28days, the incidence rate of AAD was estimated. For in vitro study, human umbilical vein endothelial cells (HUVEC) were treated by sunitinib for 24 hours. Then, mRNA expressions of intracellular cell adhesion molecule-1 (ICAM-1) and endothelin-1 (ET-1) were measured.  

Sunitinib increased systolic blood pressure (182 mmHg vs 288 mmHg with sunitinib ; p＜0.01) and the incidence of AAD (40% vs 59% with sunitinib; p=0.26). Moreover, sunitinib increased mRNA expressions of ICAM-1 and ET-1 in HUVEC. These results suggested that VEGF inhibitors induced high blood pressure and developed AAD via endothelial damage.

Pulmonary arterial hypertension (PAH) is a severe and progressive disease that causes right heart failure. The pathogenesis of PAH is generally characterized by persistent high pulmonary arterial resistance and pulmonary arterial remodeling. In this study, we investigated the effects of Eucommia ulmoides oliver leaf extract (ELE) on hypoxia-induced PAH in mice. We observed that Eucommia ulmoides oliver leaf extract (ELE) improve right ventricular systolic pressure (RVSP) and pulmonary vessel muscularization. To identify an active ingredient, geniposidic acid (～1 mg/kg, ～5 mg/kg), a major component of ELE, were orally administered to C57BL/6J mice during exposure to hypoxia for 4 weeks. Geniposidic acid significantly suppressed the elevation of RVSP in hypoxia-induced PAH mice. In addition, hypoxia-induced pulmonary arterial muscularization was slightly attenuated in geniposidic acid-treated mice. In human pulmonary artery smooth muscle cells (HPASMC), endothelin-1-induced intracellular Ca²⁺ elevation was attenuated by geniposidic acid (200 µM). Furthermore, geniposidic acid (50 – 200 µM) increased the maximal respiration in HPASMC. These findings suggest that geniposidic acid may be active ingredient of ELE which effectively improve the development of hypoxia-induced PAH by preventing the vascular remodeling and mitochondrial dysfunction of pulmonary artery.

【Introduction】Tyrosine kinase inhibitors (TKIs) have improved the survival of patients with various types of cancer. Growing evidence suggest that cancer therapy-related cardiotoxicity has become important as the serious adverse event associated with many TKIs. Epidermal growth factor receptor-TKI (EGFR-TKI), which has demonstrated efficacy in patients with non-small-cell lung cancer, has been reported to have a risk of cardiac dysfunction. However, cardiotoxicity of EGFR-TKIs has not been fully understood. Here we evaluated the effects of EGFR-TKIs on contractility using human iPS cell-derived cardiomyocytes (hiPSC-CMs).

【Methods】We used iCell cardiomyocyte 2.0 (FCDI). Motion analyses were performed using a cell motion imaging system (SI8000, Sony). Real-world pharmacovigilance data were analyzed by a reporting odds ratio from FDA Adverse Event Reporting System (FAERS).

【Results】We found that several EGFR-TKI decreased contraction velocity in a concentration-dependent manner, while other EGFR-TKIs did not. To confirm the in vitro data, we analyzed the cardiotoxicity risk of EGFR-TKIs by the real-world pharmacovigilance data from FAERS. EGFR-TKI, which decreased contraction velocity in hiPSC-CMs, was significantly associated with cardiac failure and decreased ejection fraction.

【Conclusion】Thus, contractile analysis of hiPSC-CMs would be useful to assess TKI-induced cardiac dysfunction in human. We are planning to evaluate other types of TKIs with hiPSC-CMs.

In many cardiovascular diseases, differences between women and men have been well described in epidemiology, pathophysiology, clinical manifestations, treatment efficacy, and outcomes, and have attracted attention as an aspect of personalized medicine. Recently, the use of induced pluripotent stem cells (iPS) cells to reflect individual differences in the drug discovery and the toxicological assay has been attracting attention, but a screening assay system for human cells to evaluate sex differences has not yet been established. Thus, we here propose to develop an in vitro system using iPS cell lines derived from a pair of dizygotic twins. We report the progress of three pairs of clones selected for future sex-differences analysis; no sex differences were observed in the expression of the three germline differentiation markers. As a next step, we plan to examine sex differences in gene expression in twin pairs of undifferentiated and differentiated cardiomyocytes to obtain data that will form the basis for future functional analysis of iPS cell-derived cardiomyocytes, including cardiotoxicity assessment.

Non-alcoholic steatohepatitis (NASH) is a disease in which fatty liver develops independently of alcohol intake and progresses to cirrhosis and liver cancer. Currently, no effective treatment for NASH has been found, and new approaches to elucidate the pathogenesis of the disease are needed. It's also been suggested that NASH is associated with functional abnormalities in mitochondria, which are involved in lipid metabolism. Our laboratory has successfully established liver organoids from NASH model mice that can reproduce the fibrotic pathology of NASH, and electron microscopic images of NASH liver organoids showed lipid accumulation, mitochondrial deformation, and aggregation compared to normal liver organoids. Therefore, we analyzed the morphological changes and expression levels of fibrosis-related markers in NASH liver organoids upon treatment with Mdivi-1, a mitochondrial mitogen (DRP1) inhibitor. The results showed that Mdivi-1 suppressed the expression of dendritic morphology in NASH liver organoids and decreased the mRNA expression levels of Collagen-I and α-SMA. In addition, when Mdivi-1 was administered long-term to mice fed a NASH-inducing diet, improvement of fatty liver was observed compared to the solvent-fed group. These results suggest that Mdivi-1 may be useful as a therapeutic agent to improve NASH pathology.

Immunoglobulin (IgG) therapy is a strategy for treatment of autoimmune, immunodeficiency and acute infectious diseases. Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare and refractory autoimmune disorder of the peripheral nervous system, characterized by symmetric weakness, impaired sensation and damaged myelin (demyelination). Although intravenous immunoglobulin (IVIg) preparation is used for the therapy, the mechanism of this therapy on demyelination has not been understood. In this study, we examined the effect of human IgG on the lysolecithin-induced demyelination in the mouse sciatic nerve.

Lysolecithin was injected into sciatic nerves of the ICR mice (day 0) to induce demyelination and 20 mg (i.v., day 1) and 10 mg (i.p., day 3) of IVIg preparation or the same volumes of saline for control group were administered. Demyelination area and infiltrated macrophages were evaluated with the longitudinal sciatic nerve sections on the day 7 and 14 by immunostaining. The demyelination areas of the IVIg-treated group were significantly less than those of the control group. CD68⁺ macrophages infiltrated in the lesions and CD68⁺ CD206⁺ macrophages were more prominent in IVIg-treated group.

The results suggest that IgG therapy decreased demyelination areas possibly through the accumulation of M2-type macrophages.

The prevention of oxaliplatin-induced peripheral neuropathy (OIPN) by thrombomodulin alfa (TMα) involves thrombin-dependent activation of protein C (PC) and thrombin-activatable fibrinolysis inhibitor (TAFI), in addition to inactivation of high mobility group box 1 (HMGB1). We have demonstrated that complement C5a, degradable by activated TAFI (TAFIa), known as carboxypeptidase B (CPB), is involved in OIPN development. In the present study, we examined the effect of APC on OIPN as well as surgically induced neuropathic pain in mice, and asked whether proteinase-activated receptor 1 (PAR1) would participate in the effects of APC in those neuropathic pain models, given that APC is an unbiased or biased agonist of PAR1. The OIPN in mice was prevented fully by TMα, an anti-HMGB1-neutralizing antibody (HAb) or TAFIa/CPB, and, to a lesser extent, by APC. Vorapaxar, a PAR1 antagonist, completely and partially canceled the anti-OIPN effects of APC and TMα, respectively. Interestingly, the neuropathic allodynia caused by partial sciatic nerve ligation was also abolished by TMα, and reduced by HAb or APC, and the effect of APC was reversed by vorapaxar. Our data suggest that PAR1 is involved in the preventive effects of APC and, in part, of TMα on OIPN and surgically induced neuropathic pain.

Recent studies indicate the importance of the signal relay from macrophages towards Schwann cells in axon regeneration after peripheral nerve injury. However, the molecular mechanisms underlying axon regeneration via macrophage-Schwann cell communication remain largely unclear. Here, we explored macrophage-derived molecules relevant to axon regeneration. After inferior alveolar nerve transection (IANX), the rats showed hypoesthesia in the lower lip, which was recovered from 10 days after IANX by an intrinsic regeneration capacity. In contrast, macrophage ablation caused delayed nerve regrowth. Furthermore, c-Jun-positive Schwann cells, a repair phenotype, disappeared after the removal of macrophages. Cathepsin S (CTSS) from macrophages promoted recovery from hypoesthesia and cleaved ephrin B2 on fibroblasts. EphB2, a receptor of ephrin B2, was expressed in Schwann cells. Accelerated recovery from hypoesthesia after IANX following CTSS treatment was prevented by neutralization of ephrin B2. These results suggest that CTSS from macrophages liberates ephrin B2 which in turn facilitates axon regeneration in the orofacial regions. Our results lead to the development of novel therapeutics for hypoesthesia caused by nerve injury targeting CTSS.

Background: Cell-free DNA (cf-DNA) is known to be released from injured cells and act as a critical activator of inflammation and the immune system. Patients with COVID-19 could develop respiratory failure and therefore require oxygen therapy. In this study, we hypothesized that circulating cf-DNA level could reflect the severity of COVID-19.

Methods: Analyses of cf-DNA levels were performed on serum samples from 95 hospitalized-patients with confirmed COVID-19 at Showa University Hospital (Tokyo, Japan). Cf-DNA levels were assessed by measuring the copy number of mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) using quantitative real-time PCR.

Results: Patients were grouped into Moderate, Severe, and Critical using the severity criteria by the National Institutes of Health in U.S.A. There was no significant difference on both cf-DNA levels between Moderate and Severe groups, and between Severe and Critical groups. Meanwhile, both of the levels were significantly higher in Critical group than Moderate group. Patients were also grouped by their respiratory treatment. Both cf-DNA levels significantly increased in patients with oxygen-supplementation and patients with intubation, compared to those with no oxygen supplementation and with non-intubation, respectively. There was negative association between oxygen saturation (SpO₂) and cf-nDNA levels, not cf-mtDNA.

Conclusion: These results suggest that serum cf-DNA could serve as a useful biomarker to help determining therapeutic management for respiratory failure in COVID-19.

AKI is considered as a "curable disease", but recent epidemiological studies and meta-analysis have revealed that AKI is a risk factor for CKD. Therefore, it is necessary to establish a treatment that can control the AKI to CKD transition. We have studied mild electrical stimulation (MES) and heat shock (HS; 42℃) that promotes effective biological responses. Interestingly, in adriamycin (ADR)-induced nephrotic syndrome (NS) mouse model, MES+HS significantly suppressed albuminuria and proteinuria, which are characteristics of NS. We also investigated the effects of MES+HS on AKI to CKD transition in a mouse model of bilateral ischemia reperfusion injury (Bi-IRI). The renal function of Bi-IRI mouse model was rapidly decreased and then recovered over time. However, tubular damage, inflammation and fibrosis were observed even after recovery of renal function. MES+HS promoted the recovery of renal function in this model. Moreover, MES+HS significantly suppressed tubular damage, inflammation, and fibrosis, which are indicators of AKI to CKD transition on day 14 after Bi-IRI. It has been reported that a subpopulation of failed-repair proximal tubular cell (FR-PTC) emerges after AKI and is involved in the development of chronic disorders. We found that MES+HS reduced the number of Vcam1-positive tubular cells, a marker of FR-PTC, suggesting that MES+HS promotes normal tubular repair. Together, MES+HS can suppress AKI to CKD transition by regulating inflammation, fibrosis and also the emergence of FR-PTC involved in the chronicity of the renal disorder.

Post-stroke cognitive impairment (PSCI) is one of the major complications after a stroke and affects quality of life. Recently, several studies demonstrated that elevated brain proinflammatory cytokines such as TNF-α, IL-6 and IL-1β can induce cognitive impairment. However, the mechanisms underlying PSCI remain unclear. In the present study, we investigated whether post-stroke inflammasomes are involved in the development of PSCI using acetic acid-induced embolic cerebral infarct mice. Long-term learning and memory assessed by the passive avoidance test was impaired on days 7 and 14 after stroke, whereas short-term learning and memory assessed by Y-maze test showed no changes. Also, the expression of the phosphorylated AMPA receptor subunits GluR1 at serine 831 and serine 845 in the dorsal hippocampus were significantly decreased. Under these conditions, inflammasome-related proteins, including caspase-1, ASC/TMS1, IL-1β, TNF-α and IL-18, were significantly increased in the dorsal hippocampus 14 days after stroke. The present findings suggest that a decrease in phosphorylated GluR1 at ser831 and ser845 via the inflammasome activation pathway in the dorsal hippocampus may be involved in the development of learning and memory impairment after embolic stroke.

The basolateral amygdala (BLA) shows firing activity synchronized with neocortex slow oscillations, a form of <1-Hz oscillations that occur dominantly during slow-wave sleep or under anesthesia and is believed to contribute to the formation of emotional memories. Despite its importance for elucidating the neural circuits involved in the formation of emotional memories, the mechanism of the synchronization has remained unclear, mainly because it is difficult to record neuronal membrane potentials in deep brain regions, such as the BLA. We recorded membrane potentials of BLA neurons using a new method that enables whole-cell recording from deep brain regions in vivo. We found that BLA neurons transiently depolarized at late active phases in the slow oscillations. To determine the neural source of the depolarization of BLA neuros, we focused on the medial prefrontal cortex, whose axons projecting to the BLA is known to contribute to the formation of fear memories, as a candidate region that depolarizes BLA neurons during slow oscillations. BLA-projecting neurons were retrogradely labeled with channelrhodopsin-2 using retrograde adeno associated virus, and their firing activity was recorded using an opto-tagging method. These results provide insight into the neural mechanism that synchronizes the slow oscillations between the BLA and the neocortex and lead to the elucidation of the mechanism underlying the formation of emotional memories.

(Background and purpose)

Niemann-Pick disease type C (NPC) is a genetic disorder in which patient cells have endosomal/lysosomal accumulation of cholesterol.  No approved drug improves cholesterol accumulation, and the creation of new therapeutic drugs are desired. Currently, there are many relationships between sphingolipids and NPC have been reported. This study investigated the relationship between NPC and ceramide-1-phosphate, produced by phosphorylating ceramide, using NPC1-null mice.

(Results and discussion)

NPC is characterized by clinically affecting the brain and liver; premature death invariably results.

We generated double-knockout (DKO) mice lacking NPC1 and CerK and compared the phenotypes of NPC mice and DKO mice in these tissues. In the brain, cholesterol accumulation and Purkinje cell survival were improved in DKO mice compared with those in NPC1-null mice. In the liver, cholesterol accumulation and liver disorder were improved in DKO mice compared with those in NPC1-null mice. Administration of a CerK inhibitor to NPC1-null mice delayed the onset of clinical signs and prolonged the lifespan. These results suggest that CerK may be helpful as a novel therapeutic target for NPC.

Neuropathic pain (NP) is frequently accompanied by anxiodepressive-like behaviors, yet the mechanisms remain unclear. Mitochondrial dysfunction induces neuroinflammation and has been implicated in various neurological diseases, including depression. However, the relationship between mitochondrial dysfunction and anxiodepressive-like behaviors in the NP state is unclear. The current study examined whether mitochondrial dysfunction is involved in anxiodepressive-like behaviors in mice with NP. NP was induced by partial sciatic nerve ligation (PSNL) of male ddY mice. Anxiodepressive-like behaviors were evaluated by forced swim test, social interaction test, and novelty suppressed feeding test. Mitochondrial dysfunction was assessed by quantifying mitochondrial DNA in the cytoplasmic fraction. The expression of type Ⅰ interferon mRNA was analyzed by real-time PCR. Curcumin was orally administered to inhibit mitochondrial dysfunction. PSNL induced anxiodepressive-like behaviors with accompanying mitochondrial dysfunction and increase of type I interferon mRNA in the hippocampus at 8 weeks post-injury. Curcumin suppressed mitochondrial dysfunction and improved anxiodepressive-like behaviors. The current study suggests that mitochondrial dysfunction in the hippocampus could be involved in anxiodepressive-like behaviors under NP state.

Dopamine (DA) is a critical neurotransmitter which modulates motor functions, learning and motivation. Abnormal DA signaling is related to neuronal diseases such as schizophrenia (SZ) and Parkinson's disease (PD). The symptoms of SZ are classified as hyperDAergic positive symptoms and hypoDAergic negative symptoms. PD is a common progressive neurodegenerative disorder characterized by the loss and degradation of DAergic neurons. Current drugs poorly treat both diseases.

Ceramide kinase (CerK) is an enzyme which phosphorylates ceramide, a central metabolite of sphingolipids to produce ceramide-1-phosphate (C1P). CerK/C1P pathway is reported to be involved in extracellular homeostasis of some neurotransmitters in vitro, however, the involvement with SZ or PD remains unclear.

In this study, we created SZ and PD model in mice genetically deleted Cerk and compared their phenotypes with wild-type mice. We found that Cerk deficiency exacerbated the positive symptoms of SZ partly due to an increase in the extracellular DA levels. In contrast, negative symptoms of SZ and motor dysfunction of PD were partly improved by Cerk deficiency.

These results suggest that CerK may be a new therapeutic target for hypoDAergic diseases such as negative symptoms of SZ and PD by regulating extracellular DA levels.

Although parenting behavior is necessary for development of infants, underlying neural mechanisms remain unclear. The amygdalohippocampal area (AHi) neurons projecting to the medial preoptic area (MPOA), a key region for parenting, were shown to be activated by both parenting and aggression toward pups using retrograde tracer and c-Fos immunostaining. We labeled MPOA-projecting AHi neurons in a projection-specific and activity-dependent manner using a retrograde adeno-associated virus vector expressing Cre recombinase activity-dependently and mice expressing tdTomato Cre-dependently. As a result, we observed the presence of two functionally distinct subpopulations of parenting and aggressive response neurons.

We next performed scRNA-seq to determine whether the two populations were molecularly distinct and found 395 genes exhibiting higher expression levels in the parenting subpopulation than in the aggression subpopulation, and 755 genes showing the opposite pattern. Among them, we focused on 5-HT7 receptor because of high expression of Htr7 in the parenting subpopulation. Intraperitoneal administration of LP44, 5-HT7 receptor agonist, resulted in activation of parenting-labeled, but not aggression-labeled, neurons. Finally, microinjection of LP44 into the AHi 15 min before behavioral test promoted parenting behavior.

In conclusion, we identified distinct subpopulations of MPOA-projecting AHi neurons encoding parenting and aggressive behaviors toward pups. The results demonstrated that parenting AHi neurons expressed 5-HT7 receptors and were activated by administration of 5-HT7 receptor agonist, which suppressed aggressive behavior toward pups.

Dendritic spine morphological changes occur in the brain after cerebral ischemia. Arcadlin, a non-clustered protocadherin δ2, is induced in a neuronal activity and reduces the dendritic spine density via endocytosis of N-cadherin. Cerebral ischemia induces neuronal activity but the expression of Arcadlin and its role in the ischemic brain are not clear. In this study, we analyzed Arcadlin expression and dendritic spine changes after cerebral ischemia using a highly reproducible mouse model of middle cerebral artery occlusion (MCAO). We found that Arcadlin mRNA was significantly upregulated in the hippocampal dentate gyrus (DG) at 4 hours after MCAO. Dendritic spine density in the ipsilateral DG was lower than in sham mice. These results suggest that Arcadlin may be involved in the reduction in the dendritic spine density. We are performing the comparison of dendritic spine density between MCAO and Sham using Arcadlin KO mice.

Arcadlin (Acad/Protocadherin-8) is a non-clustered d2-protocadherin of the cadherin superfamily. Acad is induced quickly by neural activity and is known as a molecule that reduces spine density in the hippocampal neurons in vivo and in vitro. Pyramidal neurons in the layer II of the piriform cortex strongly expresses Acad mRNA. The piriform cortex is often kindled electrically for seizure generations. We hypothesize that the neural activity of the piriform neurons induces Arcadlin, which in turn modulates their spine morphology. In order to investigate this possibility, we examined the dendritic morphology of the pyramidal neurons in the piriform cortex in Acad^−/− mice. Contrary to our expectation, Acad^−/− mice showed a lower spine density than WT mice in the pyramidal cells of piriform cortex. The change in spine density was most obviously observed in thin spines, and in the dendritic zone distal to the cell body. The data suggest that Acad does not suppress the piriform pyramidal cells at least in unkindled status. We will further examine whether the induced Acad suppresses the piriform pyramidal cells under kindled condition.

Diseases with peripheral inflammation, such as sepsis and peritonitis, are associated with an increased risk of central nervous system diseases. Injection of lipopolysaccharide (LPS) into mice has been widely used as a disease model for peripheral inflammation and exhibit cognitive dysfunction. Although glial cells have been implicated in the pathogenesis of cognitive dysfunction, the detailed mechanisms remain unclear, and most studies have been conducted in young or old mice. In this study, we examined the effects of LPS on short-term memory in adult mice to elucidate the mechanisms of pathogenesis.

C57BL/6N mice (male, 11-13 weeks old) were injected with LPS (3 mg/kg, i.p.). At 7 days after injection, the novel object recognition test was conducted, and LPS decreased the discrimination index. At 7 days after injection, the number of c-Fos+ cells, a marker of neuronal activation, decreased in the hippocampal CA1 region, and the percentage of immature spines detected by Golgi-Cox staining was increased. The gene expression of several inflammatory factors peaked at 1-3 days after LPS injection and recovered to pre-injection levels at 7 days after injection.

These results indicate that the short-term memory impairment was observed at 7 days after LPS injection. Inhibition of hippocampal CA1 neuron activation and the decrease in synaptic strength due to immature spines may be involved in the short-term memory impairment.

Alzheimer's disease (AD) is a neurodegenerative disorder characterised by deposition of Aβ and hyperphosphorylated tau in the brain. In addition, hyperactivation of glial cells, cerebrovascular damage, and structure changes of white matter are also observed in AD brain. We previously found that diosgenin regenerated axons in the brain and improved memory impairment in AD model mouse (5XFAD). However, other beneficial effects of diosgenin leading to memory recovery remain unknown. This study aimed to investigate the effect of diosgenin on morphology of glial cells, blood vessels, and myelin in 5XFAD mouse brains.

Vehicle solution or 0.1μmol/kg diosgenin were orally administered to wild-type and 5XFAD mice for 14 days. Diosgenin administration significantly improved object recognition memory in 5XFAD mice. The brain slices were served for immunohistochemistry. Diosgenin administration did not remarkably influence the areas of GFAP⁺ astrocytes, Iba1⁺ microglia and CD31⁺ blood vessels at least in the prefrontal cortex, hippocampus, and perirhinal cortex in 5XFAD mice. We are currently evaluating myelin formation and neurogenesis.

This study indicated that diosgenin administration didn't provide no numerical changes of astrocytes, microglia, and blood vessels in the 5XFAD mouse brains. Therefore, we are now narrowing down the most contributing factor to memory improvement by diosgenin.

Oral administration of a food-derived amino acid ergothioneine (ERGO) enhances cognition in mice although molecular mechanisms remain unclear. Our comprehensive molecular targeting assays showed inhibition by ERGO of histamine N-methyltransferase (HNMT), a main histamine-metabolizing enzyme in the brain (J Funct Foods 95, 105165, 2022). We here attempted to characterize the inhibitory effect of ERGO on HNMT-induced methyl transfer from a methyl donor S-adenosyl-methionine (SAM) to histamine. A radioenzymatic assay using a [³H]SAM showed that mouse brain homogenate increased radioactivity of the [³H]N-methylhistamine, while incubation with ERGO or an HNMT inhibitor metoprine significantly suppressed the radioactivity, suggesting that ERGO inhibits murine brain HNMT. Lineweaver-Burk analysis using human recombinant HNMT showed that the inhibition by ERGO was competitive with histamine. Quantification of N-methylhistamine by LC-MS/MS showed that human recombinant HNMT produced N-methylhistamine in a time-dependent manner, whereas ERGO and metoprine significantly suppressed the production. We then examined the activation of microglia, immune cells in the brain, because histamine is an important molecule in the immune system. Immunochemical analysis showed that HNMT was expressed in primary cultured microglia (PCM). Exposure of PCM to ERGO or metoprine significantly increased mRNA expression of CD206, a marker for anti-inflammatory M2 microglia. Thus, the HNMT inhibition by ERGO may be associated with microglial polarization in the brain.

Itch is defined as an irritating sensation that triggers a desire to scratch. The signaling pathways of itch fall into two main categories, histaminergic and non-histaminergic pathway. Mainly, C fibers transmit itch information in both pathways. Since it is known that α₂δ subunits composing voltage-gated Ca²⁺ channels are expressed in C fibers, mirogabalin, a novel gabapentinoid that targets these subunits, may be applicable as an antipruritic drug. In this study, using male ICR mice, we showed effect of mirogabalin on scratching behavior induced by several pruritogens (histamine, chloroquine and compound 48/80). Scratching bouts increased by these pruritogens are decreased by oral administration of mirogabalin (10 mg/kg). The oral administration of mirogabalin (10 mg/kg) exhibited no sedation. In addition, the scratch behavior was inhibited by intracisternal injection of mirogabalin, but not local intradermal injection. These results suggest that mirogabalin is effective against itch transmitted through both histaminergic and non-histaminergic pathway and also that central nerve system, especially spinal cord, are involved in the antipruritic effect of mirogabalin.

Background: Doxorubicin (Dox), an anticancer drug, is known to induce cardiac toxicity by causing mitochondrial dysfunction. Although CaMKII and its phosphorylation targets, Drp1 to control mitochondrial fission and MCU to control mitochondrial Ca²+ uptake, regulate mitochondrial homeostasis, the involvement of these molecules in the Dox-induced mitochondrial dysfunction remains unclear.

Method: To study the effects of Dox on mitochondrial homeostasis, we evaluated mitochondrial membrane potential (MMP), mitophagy, and mitochondrial Ca²+ content ([Ca²+]m) in H9C2 cells with the following fluorescent dyes, JC-1, Mtphagy, and Rhod2-AM, respectively. To examine the activating effect of Dox on CaMKII, we evaluated the phosphorylation levels of CaMKII by western blotting. To test the involvement of CaMKII, Drp1, and MCU in the Dox-induced mitochondrial dysfunction, the specific inhibitors, KN-93, Mdivi-1, and Ru360, respectively, were used.

Result: Dox treatment dose-dependently reduced MMP and increased the number of cells with mitophagy and [Ca²+]m (p<0.05 in all). Dox treatment significantly increased the phosphorylation levels of CaMKII (p<0.05). The inhibition of CaMKII suppressed the effects of Dox on the MMP and the mitophagy (p<0.05), but not on [Ca2+]m. Contrarily, the inhibition of Drp1 and MCU failed to suppress the decrease in MMP by Dox. Similarly, the inhibition of Drp1 did not reverse the increase in mitophagy by Dox, nor did the inhibition of MCU suppress the elevation of [Ca²+]m by Dox.

Conclusion: These results suggested that activated CaMKII, but not Drp1 and MCU, is involved in the impairment of MMP leading to Dox-induced mitochondrial dysfunction and that the excessive fission by Drp1 and the increased uptake [Ca²+]m by MCU are not the mechanism for the Dox-induced MMP reduction.

Atherosclerotic plaques are formed by the accumulation of foamy macrophages, which phagocytose oxidized low-density lipoprotein (LDL) in the intima of blood vessels. Although bioimaging to detect the accumulation of foamy macrophages is thought to be an effective tool for the prevention and treatment of atherosclerosis, it has not yet been established. In this study, we examined the bioimaging of atherosclerotic plaques using human oxidized LDL (oxLDL) labeled with ¹²⁵I in apolipoprotein E knockout (ApoE-KO) mice, a mouse model of atherosclerosis. Mouse bone marrow-derived macrophages markedly phagocytosed oxLDL but not intact LDL (LDL). The Oil Red O staining revealed that massive amounts of atherosclerosis plaques were formed in the aortic arch and aortic valve in ApoE-KO mice. In the study of single photon emission computed tomography (SPECT), distinct signals were detected in the aorta of ApoE-KO mice treated with ¹²⁵I-labeled oxLDL but not with ¹²⁵I-labeled LDL. The local distribution of radioactivity was also detected by autoradiography. We further confirmed the uptake of DiI-labeled oxLDL by macrophages accumulated in atherosclerotic plaques in ApoE-KO mice. A possibility is suggested that bioimaging for the diagnosis of atherosclerosis could be developed with the strategies such as application of radioisotope-labeled oxLDL.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs) are increasingly being used for cardiac safety evaluation, disease modeling, and regenerative medicine. To date, the majority of cardiotoxicity studies have examined the acute drug effects. However, these studies lack information on the chronic effects of cardiotoxic compounds. Here, motion vector prediction (MVP) method was employed to invasively quantify contractile function over 10 days, then to test whether nobiletin has a protective effect against thecardiotoxicity of 8-days exposure to doxorubicin and erlotinib. The MVP method showed that doxorubicin (0.1 - 0.3 μM) significantly reduced contractility compared to erlotinib (0.3 - 3 μM), which has no cardiotoxicity, when administered for more than 5 days, and that the simultaneous addition of 0.03 μM nobiletin significantly reduced this cardiac depression by doxorubicin (0.1 - 0.3 μM). These results suggest that nobiletin is protective against the cardiotoxic effect of doxorubicin, and are promising for future drug discovery applications.

Accumulation and aggregation of α-synuclein (α-Syn) are hallmarks of α-synucleinopathies such as dementia with Lewy bodies. Therefore, aggregation of α-Syn is considered a priority target for drug development, and aggregation inhibitors are expected to reduce α-Syn toxicity and serve as therapeutic agents. Here, we report that certain lysophospholipids (LPLs) species behave as inhibitors for α-Syn aggregation. The LPLs are small bioactive lipid molecules characterized by having a single carbon chain and a polar head group. The LPLs we used were extracted from Porcine Liver Decomposition Product (PLDP) which was previously reported to enhance cognitive function in healthy older adults. We found that the LPLs extracted from PLDP (PEL) reduced α-Syn aggregation in cellular model. Especially, four species of LPLs contained in PEL strongly inhibit α-Syn aggregation. Furthermore, we revealed that PEL increased normal cell viability in SK-N-SH cells. Finally we approached the mechanism of the LPLs' inhibitory effect forα-Syn aggregation using in vitro assay and evaluated influences to various cellular functions known to be disordered in lesion. Taken all together, these studies indicate that the LPLs would be beneficial as a possible therapeutic target in the treatment of α-synucleinopathies.

Astrocytes constitute about 20-40% of glial cells, making up the central nervous system (CNS). In the CNS, astrocytes supply neurons with nutritional factors and maintain homeostasis of the extracellular environment through the uptake and efflux of neurotransmitters. In neurodegenerative diseases, astrocytes change their morphology to the activated state and release inflammatory cytokines, which induce CNS inflammation and aggravate neurodegenerative diseases. Sphingolipids, one of the lipids, have been reported as a molecule associated with astrocyte activation, but their involvement remains unclear. In this study, we evaluated astrocyte activity by changing the levels of sphingomyelin (SM), one of the most abundant sphingolipids, using human astrocyte/conditionally immortalized clone 35 (HASTR/ci35). Reduction of SM levels by knockdown of sphingomyelin synthase 1 and/or 2 in HASTR/ci35 attenuated HASTR/ci35 activation by treatment of inflammatory cytokines. Furthermore, selectively reducing SM levels by knockdown of ceramide transport protein also attenuated the activation of HASTR/ci35. On the other hand, increasing the levels of SM by inhibition of neutral sphingomyelinase or addition of SM exogenously promoted activation of HASTR/ci35 by treatment of inflammatory cytokines. These results suggest that SM positively regulates astrocyte activation. Thus, regulating SM levels may provide a therapeutic target for astrocyte-induced CNS inflammation and a new approach to treating neurodegenerative diseases.

In Alzheimer's disease (AD), acetylcholinergic (ACh) neurons are impaired at early pathological stage of AD, and amyloid β (Aβ) oligomers are thought to be a crucial molecule for triggering neurodegenerative processes in AD. In the present study, we first established an in vitro Aβ oligomer-induced neuronal cell death model using human induced pluripotent stem cell (iPSC)-derived ACh neurons and O-acyl isopeptide of Aβ, which reverts to natural form of Aβ under the neutral pH conditions. In the processes of neurodegeneration in this model, Aβ was tightly attached dendrites and mitochondrial dysfunction was induced. We next identified an Aβ-binding low molecular weight compound, plantainoside B, from the herbal extract of Bacopa monniera, which is used for memory enhancement in Ayurvedic medicine. Plantainoside B attenuated mitochondrial dysfunction and exert neuroprotection against Aβ neurotoxicity. Moreover, the ¹²⁵I-labeled plantainoside B showed a high affinity to brain sections obtained from a model mouse of Aβ plaque formation and Aβ oligomers in gel-loading experiments. Results indicate a possibility for the development of neurotheranostics approach for the strategy of AD treatment.

Spinocerebellar ataxia (SCA) is a group of autosomal-dominantly inherited ataxia and classified into SCA1-49 by the difference of causal genes, whose mutations include polyglutamine (polyQ)-expanded and missense mutations. Purkinje cells (PCs) are neurons with highly developed dendrites and important for cerebellar functions. We have previously revealed that impaired dendritic development is observed in primary cultured PCs expressing missense mutant SCA14- and SCA21-causing proteins. We assume that various SCA-causing proteins commonly impairs dendritic development in cultured PCs. In the present study, we expressed polyQ-expanded (SCA1, 3 and 6) and missense (SCA34, 38, 41) mutant SCA-causing proteins in primary cultured PCs and evaluated their dendritic development. Cerebellar primary cultures were prepared from E16 embryos of Wistar rats and cultured for 3 weeks. SCA-causing proteins were expressed using adeno-associated viral vector. Compared with wild-type proteins, all 6 SCA-causing proteins, including polyQ-expanded and missense mutants, impaired dendritic development of primary cultured PCs. These findings indicates that impaired dendritic development of cultured PCs is induced by various SCA-causing proteins and would be a common in vitro phenotype of SCA and available for the exploration of novel SCA therapeutics.

Liver fibrosis is a significant consequence of chronic liver diseases, where excess deposition of extracellular matrix is caused by the activation of hepatic stellate cells (HSCs). The suppression of HSC activation is therefore regarded as a therapeutic target of liver fibrosis. The present study investigated the involvement of protein arginine methyltransferase 5 (PRMT5), which mediates genome organization and cell cycle regulation, in HSC activation. LX-2 cells, a human HSC cell line, were treated with TGF- β1 for 48 h in the presence of PRMT5 inhibitors (EPZ015666 and JNJ64619178). The expression of α-smooth muscle actin (α-SMA) and type I collagen α1 (COL1A1), activated HSC markers, were markedly increased by the TGF-β1 treatment. PRMT5 inhibitors suppressed the increased expression of α-SMA and COL1A1 in a concentration-dependent manner. Knockdown of PRMT5 also suppressed the TGF-β1-induced COL1A1 expression in LX-2 cells. RNA-sequencing analysis showed that GO terms related to ECM production and SMAD signaling were enriched with RNA of LX-2 cells treated with the PRMT5 inhibitor JNJ64619178. These results suggest that PRMT5 promotes HSC activation, possibly depending on the SMAD signaling pathway, and therefore might be a target for the prevention and treatment of liver fibrosis.

Currently, there is no effective treatment for liver fibrosis. In vitro studies on the activation of hepatic stellate cells (HSCs), which is responsible for liver fibrosis, have been used as a drug screening system for it. However, even if some drug shows an inhibitory effect on HSC activation, its anti-fibrotic effect is required to be confirmed in liver fibrosis animal models, which further requires a lot of time and cost. In the present study, we tried to establish an ex vivo model of liver fibrosis using precision-cut liver slices (PCLSs) to solve such problems. PCLSs of 250 µm thickness were prepared from male C57BL/6J mice using a vibratome and cultured in RPMI medium in a 5% CO₂ incubator. Although cellular ATP content was decreased on day 1 compared to day 0, it was then maintained until day 5, suggesting that the ex vivo model is viable for at least 5 days. Treatment with Et-OH (50, 100 mM), one of the liver injury stimuli, for 5 days increased mRNA expression of Acta2 and Col1a1, liver fibrosis markers, in PCLSs. DIF-1 (50, 100 µM), which has an anti-fibrotic effect, significantly suppressed the Et-OH-induced increases in the markers. These results suggest that the ex vivo model using PCLSs is useful as a drug screening system for the development of drugs for treatment of liver fibrosis.

IL-33 contributes to the pathogenesis of allergic diseases. Upon the binding ofIL-33 to a membrane receptor ST2, IL-1 receptor accessory protein (IL-1RAcP) isrecruited to form a heterodimeric receptor complex which transmits a signal. Onthe other hand, a soluble form of ST2 acts as an endogenous inhibitor of IL-33signaling. Here, we aimed to develop the biologics inhibiting IL-33 signaling.First, we generated IL-33 reporter cell lines, in which IL-33 stimulationinduced NFkB-driven expression of DsRed. We then generated IL-33trap-Fc which iscomposed of extracellular domains of IL-1RAcP and ST2 fused to the Fc portion ofhuman IgG. Notably, IL-33trap-Fc more strongly suppressed IL-33-stimulated DsRedexpression in the reporter cells than ST2-Fc which is composed of anextracellular domain of ST2 fused to human IgG Fc. Consistently, IL-33trap-Fcremarkably inhibited IL-33-stimulated IL-6 production of bone marrow-derivedmast cells as compared with ST2-Fc. Moreover, intraperitoneal administration ofIL-33trap-Fc efficiently inhibited IL-33-stimulated eosinophil accumulation invivo. Taken together, these results indicated that IL-33trap-Fc was effective ininhibiting IL-33 signaling. We also attempt to generate the biologics that bindto ST2 but fail to recruit IL-1RAcP, thereby blocking IL-33 signaling.

[Aim] Lenvatinib (LEN), an oral tyrosine kinase inhibitor, is widely used to treat several types of advanced cancers but often causes muscular adverse reactions. Our previous study revealed that LEN reduces L-carnitine (L-CAR) content, expression of carnitine-related genes, and mitochondrial function in the skeletal muscle. Therefore, the present study aimed to investigate whether L-CAR supplementation prevents LEN-induced muscle impairment without affecting its anti-angiogenesis effect.

[Methods] Eight-week-old male Wistar rats were divided into four groups and administrated orally once daily for 2 weeks with vehicle, LEN (2 mg/kg/day), LEN + L-CAR (150 mg/kg/day), or LEN + L-CAR (300 mg/kg). In the in vitro studies, differentiated C2C12 myocytes, HUVECs, and mouse aorta were treated with LEN (0.1 and/or 1 μM) and L-CAR (1.6, 6.4, and 25.6 mM).

[Results] L-CAR supplementation significantly attenuated LEN-induced deleterious effects on L-CAR content, expression of carnitine-related (OCTN2, CPT1, CACT, and CPT2) and OXPHOS genes in the skeletal muscle of rats. In addition, L-CAR prevented LEN-induced reductions in mitochondrial function (ATP content and membrane potential) in C2C12 myocytes. Furthermore, L-CAR did not affect the anti-angiogenesis action of LEN assessed by the tube formation and ring assays.

[Conclusion] These results suggest that L-CAR supplementation can alleviate the adverse reactions of LEN in the skeletal muscle without reducing its antineoplastic effect.

We report a mutagenesis strategy that can effectively increase the dissociation rate of antibodies by orders of magnitude without compromising the binding specificity. Single-molecule localization microscopy greatly surpasses the diffraction limit of conventional optical microscopy. The imaging fidelity and labelling density, however, are limited by spatial interference between bulky antibodies in a confined resolved area. IRIS has overcome the problem using exchangeable probes that transiently bind to endogenous targets. In our previous research, generation of fast-dissociating IRIS probes has been challenging. In the present study, we have developed a new mutagenesis strategy that make it feasible to generate IRIS probes from the repository of off-the-shelf antibodies. We successfully generated dozens of IRIS probes and demonstrate multiplexed localization of endogenous proteins in primary neurons that visualizes small synaptic connections. Our fast-dissociating probes achieved 4-fold higher label density than conventional super-resolution approaches. Thus, IRIS could visualize the feature of synaptic components with higher fidelity. In addition, the mutagenesis strategy will provide more applications for high affinity antibodies developed in pharmaceutical research, such as super-resolution imaging based disease diagnosis and biomarker identification.

Objective: Lupus nephritis is a typical clinical manifestation of SLE. We previously reported that LPA treatment improves depressive-like behavior and microglial activation in MRL/lpr mice (an animal model of SLE). Thus, we examined the effects of LPA on renal function and glomerulonephritis in MRL/lpr mice. Method：18-week-old MRL+/+ mice (n=12) were treated with vehicle and 18-week-old MRL/lpr mice (n=24) were treated with vehicle or LPA (1 mg/kg) for 2 weeks. After the treatment, urine and blood samples were collected, and histological examinations were performed. Results and Discussion: The significant increases in daily urinary albumin levels in MRL/lpr mice were lost by LPA treatment. Creatinine in plasma was not significantly different between the three groups. The significant increases in plasma dsDNA antibody levels in MRL/lpr mice were lost by LPA treatment. The increases of CD68-positive cells in the glomerulus were found in MRL/lpr mice and the increases were suppressed by LPA treatment. The PAS-positive rates in MRL/lpr mice were significantly increased compared to MRL+/+ mice and the increases were significantly suppressed by LPA treatment. These results suggest that LPA treatment improves glomerulonephritis and proteinuria in MRL/lpr mice.

It is well known that chronic inflammation causes the sarcopenia. Whether the efforts to prevent skeletal muscle loss benefit the immune homeostasis remains unclear. In the present study, the efficacy of leucine supplementation on the maintenance of systemic immune function was evaluated. A sciatic nerve denervation-induced sarcopenic model was established and the volume and thickness of skeletal muscle on the hindlimb were evaluated by magnetic resonance imaging. Oral administration of leucine was carried on for 56 weeks. The skeletal muscle mass and the subsets or function of immune cells were analyzed. 

In leucine-treated sarcopenic mice, skeletal muscle mass on the hindlimb was significantly increased compared to that in non-treated sarcopenic mice. Leucine treatment repaired the mitochondria dysfunction in splenocytes from sarcopenic mice both in vitro and in vivo. In sarcopenic mice, an increase of the PD-1 expression was observed in CD4+ and CD8+ T cells. However, oral leucine administration restored the expression of PD-1 in the lymphocytes to the level of non-sarcopenic mice. 

In conclusion, the administration of leucine exhibits beneficial effects on sarcopenia and may influence the anti-cancer immune responses via adaptive immune resistance mechanism.

Mechanisms underlying development of lung fibrosis in severe asthma have been unclear. In our murine models of mild and severe asthma, lung fibrosis was significantly developed only in the severe asthma model. RNA-seq analyses revealed that not only genes of collagen type 1 but also genes of matrix metalloproteinases (MMPs) and activins, TGF-b family cytokines were found to be highly expressed in the lung of the severe asthma model compared with that of the mild model. More interestingly, pathway analyses showed that pathway of CCR5 and the ligands were up-regulated in the severe model. In this study, roles of CCR5 in the development of lung fibrosis in the severe model were analyzed. Treatment with a CCR5 antagonist, maraviroc but not dexamethasone exerted significant inhibition of the development of lung fibrosis. Interstitial macrophages (IMs) that expressed CCR5 were markedly increased in the lung, and the degree was significantly higher in the lung of severe asthma model than that of the mild asthma model. Real-time RT-PCR analyses revealed that IMs derived from the lung of severe model expressed higher mRNA levels of MMPs and activins than those of mild asthma model. From these results, it was strongly suggested that CCR5⁺ IMs were possibly involved in the development of the lung fibrosis in severe asthma.

Inflammatory diseases lead to excessive proinflammatory cytokine production (cytokine storms). The cytokine storms are highly lethal, so establishing an effective treatment is desirable. Recently, some of selective serotonin reuptake inhibitors (SSRIs), which are used to treat depression, have been reported to be effective in treating various inflammatory diseases, such as COVID-19. In this study, in order to elucidate which SSRI would be the most suitable as an anti-inflammatory drug, we investigated that the effect of 5 SSRIs on the production of inflammatory cytokine (Interleukin-6; IL-6) induced by macrophage activation induced in a Toll-like receptor (TLR)-dependent manner, and by T cell activation induced in a TLR-independent manner. In J774.1 murine macrophage cells, pretreatment with the SSRIs significantly suppressed IL-6 release induced by TLR3, TLR4, and TLR9 agonist. On the other hand, these SSRIs are also significantly suppressed IL-6 release induced by T cell activator in murine splenic lymphocytes. Our results show that fluoxetine has potent inhibitory effect on IL-6 production induced by various stimuli and low cytotoxicity among the 5 SSRIs. An examination of the structural requirements indicated that the N-methyl group of fluoxetine has a critical role in the inhibition of IL-6 production. Overall, our findings suggest that fluoxetine might be one of the preferred SSRI for further evaluation as an anti-inflammatory drug to treat cytokine storms.

Prostaglandin (PG) E₂, a bioactive lipid biosynthesized from arachidonic acid, exerts its functions through 4 cognate receptors EP1-4. Our previous studies suggested that PGE₂ attenuates antitumor immunity in the tumor microenvironment by the recruitment and activation of regulatory T cells (Tregs), which are a subset of T cells specialized in immunosuppression. However, whether such actions of PGE₂ on Treg is a direct or indirect action remains largely unknown. To address this question, we generated induced Tregs (iTregs) from purified splenic CD4 ⁺ T cells by CD3/28 stimuli in the presence of TGF-β in vitro and examined the direct effect of PGE₂. Using flow cytometry analysis, we found that the expression level of Foxp3, a master transcription factor of Treg, and 4-1bb, a coactivator of Treg, were both significantly enhanced upon PGE₂ treatment. Furthermore, these effects were notably suppressed in the presence of EP4 antagonist, suggesting that PGE₂ contributes to Treg activity through the EP4 receptor. In addition, RNA-seq revealed that not only 4-1bb, but other Treg coactivator genes such as Ctla4, Gitr, and Ox40, were greatly increased in the PGE₂ treatment group. Given that previous studies reported that high expression level of these genes in intratumoral Tregs is positively correlated to Treg immune suppression activity, we speculated that PGE₂ produced in the tumor microenvironment may directly induce the activated phenotype of intratumoral Treg through EP4 receptor.

As there are no effective treatments for muscular dystrophy (MD), identifying systemically acting small-molecule therapeutics is desirable. Tyrosine phosphorylation of β-dystroglycan, which occurs via tyrosine kinase in dystrophin-deficient muscles, has been reported to induce muscle damage, and several tyrosine kinase inhibitors have been researched as potential therapeutic agents for MD. Nilotinib, a second-generation tyrosine kinase inhibitor, was potentially effective in MD by reducing muscle fibrosis. However, there was a problem that its direct effect on satellite cells inhibited muscle differentiation. Dasatinib, a third-generation tyrosine kinase inhibitor, is also expected to be effective in MD, but its effect on muscle regeneration is unknown.

Here, we investigated the effects of dasatinib on muscle, with a focus on muscle regeneration. We administered dasatinib to mice whose tibialis anterior muscle was damaged by cardiotoxin. In the dasatinib-treated mice, abnormal myofibers were observed, and muscle regeneration may be impaired. The effect on muscle differentiation was examined using the myoblast cell line C2C12. There was abnormal cell fusion, and this abnormality differed from that previously described for nilotinib. Further research is currently underway into mechanisms causing the abnormal muscle regeneration.

The chronic hyperglycemia in type 2 diabetes causes deterioration of pancreatic β-cell dysfunction

due to decreases in insulin secretory response and β-cell mass. Nobiletin, a citrus flavonoid, has been

reported to improve hyperglycemia and insulin resistance in type 2 diabetic model mice. We

previously showed that nobiletin, a citrus flavonoid, exerts insulinotropic and anti-apoptotic effects

in pancreatic β-cells through the elevation of cAMP levels. In the present study, we investigated

mechanisms for anti-apoptotic effects of nobiletin in INS-1 cells, a rat β-cell line. Endoplasmic

reticulum stress was induced by thapsigargin, tunicamycin, or chronic high glucose exposure. The

expression of apoptosis-related proteins in INS-1 cells was analyzed by Western blotting. Nobiletin

significantly suppressed the elevation of cleaved caspase-3 expression induced by these proapoptotic

stimulations. The expression of thioredoxin interacting protein (TXNIP), a regulator of cellular

oxidative stress, was also suppressed by nobiletin treatment. Nobiletin slightly restored the decrease

in phosphorylated Akt levels induced by thapsigargin or tunicamycin treatment. These results

suggest that nobiletin suppresses apoptosis induced by endoplasmic reticulum stress via the

degradation of TXNIP, which might be mediated by Akt.

Acute kidney injury (AKI) is a catastrophic disease with high morbidity and mortality in hospitalized patients and contributes to the pathogenesis of chronic kidney disease. However, there are no approved effective treatment for AKI. There are many causes of AKI, including ischemia, hypoxia, and nephrotoxicity. The primary cause is ischemia reperfusion injury, due to trauma, shock, sepsis, and renal transplantation. Previous studies have shown radical scavenging activity and anti-inflammatory activity of an alkali extract of Sasa sp. The present study aimed to evaluate the efficacy of an alkali extract of Sasa sp. in AKI model mice. AKI was induced by temporary vascular clamping of the left kidney for 45 min followed by reperfusion, two weeks after removal of right kidney. We have measured the renal function using urine and serum, and morphological assessment. The levels of inflammation markers in the kidney have also been measured by ELISA. An alkali extract of Sasa sp. improved the renal function, the damage of renal tubules and exhibited anti-inflammatory in the kidney. These findings indicate that an alkali extract of Sasa sp. protects kidney in AKI model mice.

In cervical spondylotic myelopathy (CSM), degenerative changes affecting the vertebrae, intervertebral disks and ligaments compress the spinal cord. Subsequent neuronal damage leads to motor and sensory dysfunctions. Although standard treatment of CSM is surgical decompression, neurological impairment sometimes remains or recurs. Therefore, fundamental therapy that recovers neuronal damage is required.

Our previous study clarified that Cistanche tubulosa (CT) extract improved motor dysfunction in spinal cord injury. Since we supposed that the extract might be effective also for CSM, this study aimed to investigate therapeutic effects of CT extract on CSM.

30% ethanol CT extract was orally administered to CSM model mice. Motor and sensory functional changes were observed. Recovery of spinal axons were also evaluated by visualization using neuronal tracers. To clarify functional mechanism of CT extract to axons, we focused on acteoside and echinacoside that are main components in CT extract. After oral administration of CT extract, echinacoside transferred to the spinal cord and brain. Acteoside also transferred to the spinal cord. Therefore, we investigated effects of these components on primary cultured cortical and spinal neurons. Acteoside increased axonal and dendrite densities.

This study showed that acteoside transferred to the spinal cord after oral administration of CT extract, and extended axons in vitro. Experiments of CT extract on functional recovery and axonal repairing in CSM model mice are now under investigating.

Cancer cachexia affects many patients with terminal cancer. It significantly reduces their quality of life and also affects their survival rate. Biological homeostasis helps the body adapt to stimuli such as stress and environmental changes, and a breakdown of this control mechanism is thought to lead to the development of various diseases. To better understand the fluctuations of biological homeostasis in these disease pathologies, it is important to understand cellular metabolomics. In this study, we focused on metabolic changes in hypothalamic glial cells under conditions of cancer cachexia. As a result, we found that the expression of several mRNAs involved in glycolysis and the TCA cycle were markedly altered in glial cells isolated from the hypothalamic region of cancer cachexia model mice. Based on these results, we speculate that metabolic abnormalities may be induced in hypothalamic glial cells under the pathological condition of cancer cachexia. We are currently investigating the details by a comprehensive metabolomic analysis.

Chronic obstructive pulmonary disease (COPD) is the third leading cause of death in the world and is characterized by inflammation, emphysema and respiratory dysfunction. The development of radical treatment and the achievement of long-term disease management are urgent issues for COPD. Because COPD is highly complicated with lung cancer and this complication leads to poor prognosis, it is very important to elucidate its pathological mechanism and develop a novel therapeutic strategy. Previous research showed the relationship between lung cancer and COPD-derived inflammation, but not emphysema. Therefore, I selected elastase model and induced lung cancer using tobacco-specific carcinogenesis (NNK) for lung cancer initiation stage model (COPD-NNK) and lewis lung carcinoma (LLC) for lung cancer exacerbation stage (COPD-LLC). In COPD-NNK model, incidence of lung tumors was increased but COPD phenotypes were not exacerbated. Moreover, I found that α7nAChR-p-Akt pathway was activated in these tumors. Additionally, the survival rate was decreased and intratumor T cells and immune checkpoint protein PD-L1-positive macrophages were increased in COPD-LLC model. In contrast, the survival rate was increased in anti-PD-L1 antibody-treated COPD-LLC model. These results suggested that PD-L1-positive macrophages inhibited T cell activity and decreased the survival rate. In this study, I revealed changes in tumor microenvironment and exacerbation mechanism in COPD-lung cancer complication.