The unilateral peripheral injury or inflammation produces in bilateral hypersensitivity to pain in the ipsilateral and contralateral sides. This phenomenon is reported in clinical pain syndromes and in various animal pain models. However, its mechanism is not cleared. In the present study, we investigated the mechanism of this phenomenon using the inflammatory pain model and the capsaicin test. Chronic inflammatory pain was induced by injecting the complete Freund's adjuvant (CFA) and assessed the mechanical allodynia by von Frey filament test. After CFA injection, the paw withdrawal threshold in CFA injected paw reached a minimum at 3 days after CFA injection. Furthermore, in mice pretreated with CFA in the left hindpaw 3 days before, injection of low-dose (this dose did not produce significant pain-related behaviors) capsaicin in the right hindpaw induced the remarkable pain-related behaviors against capsaicin non-injected paw. This phenomenon was attenuated by the administration of TRPV₁receptor antagonist. Moreover, bilateral increase of the mRNA expression level of spinal TRPV₁receptor was observed at 3 days after CFA pretreatment. These results suggest that the bilateral activation of TRPV₁receptor under the inflammatory state induced capsaicin related pain in non-injected side.

Chemotherapy-induced peripheral neuropathy (CIPN) is a common treatment-related adverse effect often associated with several chemotherapeutic agents. Here, we describe the therapeutic and preventive effects of TRK-700 on CIPN in rat models. The therapeutic effects were evaluated by von Frey test and cold plate test. On the other hands, the preventive effects were evaluated in the same way after the repeated administration prior to the induction of CIPN. As the results, TRK-700 attenuated mechanical allodynia and cold allodynia in a rat model of CIPN by oxaliplatin. In addition, after the repeated administration prior to the induction of CIPN by oxaliplatin, paclitaxel or bortezomib, TRK-700 prevented the development of mechanical allodynia and cold allodynia (oxaliplatin only). Interestingly, the anti-allodynic effects were maintained at least 1 week after the drug withdrawal. Therefore, TRK-700 may have a therapeutic potential in the treatment of CIPN.

Lavender and rose essential oils, mixtures of plant-derived volatile organic chemicals (VOCs) obtained from the flowers, produce behavioral effects such as antianxiety-like effects in mice in the conflict tests after intraperitoneal administration. Linalool and 2-phenethyl alcohol are the major constituents of lavender and rose essential oils, respectively. Given that linalool and 2-phenethyl alcohol also produce antianxiety-like effects in the conflict tests after intraperitoneal administration, it is suggested that linalool and 2-phenethyl alcohol enter the mouse brain after intraperitoneal administration of the essential oils to produce the behavioral effects. We developed a simultaneous blood and brain microdialysis method in a free-moving mouse, and examined whether linalool and 2-phenethyl alcohol appear in the mouse brain after intraperitoneal administration of the essential oils using the microdialysis method coupled with GC/MS analysis. The solid phase micro extraction method was used to extract VOCs from the dialysate samples. Results of an intravenous injection of lucifer yellow showed that the blood brain barrier was functioning in the mice under the experimental conditions. Linalool was detected in both blood dialysate and brain dialysate samples after the administration of the lavender essential oils. 2-Phenethyl alcohol was also detected in both blood and brain samples after administration of the rose essential oil. Peak area-time profiles for linalool and 2-phenethyl alcohol were obtained. The results support the notion that linalool and 2-phenethyl alcohol enter the brain after the intraperitoneal administration of the lavender and rose essential oils.

Neurogenesis is the process by which undifferentiated progenitor cells convert into mature and functional neurons. Impairments in neurogenesis are associated with neurodevelopmental and neuropsychiatric disorders. Elucidating the molecular mechanisms underlying neurogenesis can stimulate further understanding of the pathophysiology and the discovery of novel therapeutic targets for these disorders. In this study, we performed a comparative transcriptomic analysis to reveal common targets of the proneural transcription factors, Neurog1/2 or Ascl1, during neurogenesis of human and mouse stem cells. We successfully identified C3orf70 as a novel common target of Neurog1/2 and Ascl1 during neurogenesis. Two orthologs of C3orf70 were expressed in the midbrain and hindbrain of zebrafish larvae. We generated c3orf70 knockout zebrafish using CRISPR/Cas9. The expression of the mature neuron markers elavl3 and eno2 was significantly decreased in c3orf70 knockout zebrafish. The expression of irx3b, a zebrafish ortholog of IRX3 and a midbrain/hindbrain marker, was significantly reduced in c3orf70 knockout zebrafish. Neurobehaviors related to circadian rhythm and changing light-dark conditions were significantly impaired in c3orf70 knockout zebrafish. These results suggested that C3orf70 is involved in neural and neurobehavioral development. Defects in C3orf70 may be associated with midbrain/hindbrain-related neurodevelopmental and neuropsychiatric disorders.

We discovered a novel orally-available and brain-penetrant phosphodiesterase 1 (PDE1) inhibitor, DSR-141562.  This compound has high selectivity for the PDE1 family over other PDE families and 65 off-targets.  Further, it has the preferential selectivity for predominantly brain-expressed PDE1B over other PDE1 family isoforms.  Since PDE1B is believed to regulate dopaminergic and glutamatergic signal transduction, we evaluated the effects of this compound using schizophrenia-related behavioral assays.  DSR-141562 at 3-30 mg/kg potently inhibited methamphetamine-induced locomotor hyperactivity in rats, while it had only minimal effects on the spontaneous locomotor activity.  DSR-141562 at 0.3-3 mg/kg reversed social interaction and novel object recognition deficits induced by repeated treatment with an N-methyl-D-aspartate receptor antagonist phencyclidine, in mice and rats, respectively.  In common marmosets, DSR-141562 at 3 and 30 mg/kg improved success rate in the object retrieval with detour task.  DSR-141562 at 30 and 100 mg/kg potently elevated the cGMP concentration in monkey cerebrospinal fluid, which could be used as a translational biomarker.  These results suggest that DSR-141562 would have the therapeutic potential for positive symptoms, negative symptoms and cognitive impairments in schizophrenia.

Parental care is necessary for children to grow in mammals. Because pups can obtain their energy only from milk, dams need to care their pups even under stress conditions. Dams may cope with stress in a special manner. In the present study, we examined the neuronal mechanism for stress coping in lactating female mice. The restraint stress (RS) for four hours per day for five consecutive days increased the latency to immobilization and decreased the duration of immobilization in the forced swim test. Repeated RS did not change the immobilization in forced swim test in male mice and virgin female mice. These results indicate that dams increased active coping behavior following repeated RS. We next investigated the noradrenergic neuronal pathway from the locus ceruleus (LC) to the medial prefrontal cortex (mPFC). The analysis using a retrograde neuronal tracer fluorogold indicated that the number of NA neurons activated by forced swim test was less in RS dams than that in control dams. The number of c-Fos-positive cells in the mPFC was also less in RS dams than that in control dams. These results indicate that suppression of NA neuronal pathway from the LC to the mPFC may be involved in increasing active stress coping behavior in dams.

Parental stress is one of the factors to cause child abuse. However, not all of the parents cease to raise their children under severe stress conditions. The failure of stress coping may be involved in child abuse. In this study, we investigated the neuronal mechanism for stress coping using a social stress in lactating female mice. A C57BL/6J dam was housed with a strange ICR male mouse for ten minutes at postpartum day one, followed by exposure to the male overnight with a transparent separation. After repetition with the same procedures for four consecutive days using a strange male every day, the behavioral tests were performed. The repeated social stress mildly inhibited maternal care and decreased immobility in the forced swim test, indicating that dams exposed to the social stress nurture their pups with increasing active coping behavior. We next investigated whether noradrenergic neurons of the locus coeruleus (NA-LC) involved stress coping in dams. Chemogenetical inhibition of NA-LC during social stress severely prevented maternal care and increased immobility in the forced swim test. These findings indicate that NA-LC are required for coping with social stress in lactating female mice. The failure of stress coping via NA-LC may cause neglect in child care with depression.

Orexin neurons regulate physiological functions, including not only energy homeostasis and wakefulness, but also motivated behaviors. These neurons seem to play important roles in linking metabolic need to motivated behaviors via the dopaminergic system. Recently, we developed a rat model that expresses the Cre recombinase specifically in orexin neurons. Moreover, we established the gambling test for assessing reward motivation and decision-making under conditions of uncertainty, as well as a touch-screen system for assessing reward sensitivity and craving. Here, we examined the roles of orexin neurons in reward motivation and decision-making when orexin neurons are manipulated using pharmacogenetics approaches. In the gambling test for rodents, cell-specific excitatory manipulation of orexin neurons of rats using the DREADD technology resulted in risky arm-choice. Positive, but not negative, reward prediction error may contribute to reward-based risky choice when orexin neurons are activated. The motivational values of a large reward was increased when orexin neurons were activated by the DREADD technology. In the probability reversal learning test, cell-specific excitatory manipulation of orexin neurons resulted in low performance in rats. These results suggest that activated orexin neurons affect motivational processes, and may alter strategy in reward-based choice behavior, potentially leading to an action that fails to yield rewards and detect changes in reinforcement contingencies.

Schizophrenia is a disorder that is characterized by disturbances in thinking, perception, emotions and behavior. However, there are currently no validated biomarkers for schizophrenia. CRMP2 (collapsin response mediator protein 2) is an intracellular protein that plays an important role in mediating axon guidance molecules, dendritic branching and spine formation through its phosphorylation at the serine 522 residue. Accumulating evidence suggests that CRMP2 is implicated in neuropsychiatric disorders. We examined protein levels in brain specimens of Schizophrenia and control patients. Increased levels of total CRMP2 were found. In many cases this increased CRMP2 lowered the p-CRMP2:total CRMP2 ratio. To determine whether or not CRMP2 is a potential biomarker, we established an in vitro assay system, which allows us to evaluate the amount of CRMP2 and its phosphorylated CRMP2 in human peripheral blood mononuclear lymphocytes. The absolute value of CRMP2 of schizophrenic patients was higher than that of healthy control subjects under the age of 30 years. The relative amount of phosphorylated form of CRMP2 (pS522-CRMP2) to total CRMP2 (pS522-CRMP2/CRMP2) was low in the young schizophrenia group than the age-matched control young group. These results suggest some alteration of the protein levels and/or posttranslational modification of CRMP2 from schizophrenia patients, providing CRMP2 as a potential biomarker for schizophrenia.

Fibromyalgia (FM) is a common and chronic syndrome that causes bodily pain and mental distress. The cause of FM is largely unknown, but it is expected that changes in dopamine (DA) transmission may be involved in these symptoms. In the present study, to investigate the pathophysiological mechanisms of FM, we applied for the stem cell technology using induced pluripotent stem cells (iPSCs). We differentiated iPSCs derived from FM patients into DA neurons following previous methods. Most of the differentiated cells derived from control and FM-specific iPSCs were labeled by antibodies to βⅢ-tubulin (a neuron-specific marker) and tyrosine hydroxylase (TH) (a DA neuronal marker). Under the present condition, we performed the gene expression analysis of DA neurons derived from control or FM-specific iPSCs. We focused on the possible changes in the expression of DA synthesis or metabolism-related genes among the mRNAs changed in FM patients. With no change in tyrosine hydroxylase (TH), dopa decarboxylase (DDC) and monoamine oxidase B (MAOB), the expression of catechol-O-methyltransferase (COMT) was significantly increased in FM-specific iPSC-derived DA neurons. These finding indicate that alterations of DA metabolism in FM-derived DA neurons may contribute, at least in part, to psychiatric and pain symptoms in FM patients.

[Introduction] Early postnatal period is sensitive window for the development of neural circuits and brain functions. Thus, stressful experience in early life increases risks of various psychiatric symptoms including social dysfunction. However, mechanisms how early life stress disrupt the development of social circuits in brain were less understood. In this study, to address this issue, we examined the effects of adolescent social isolation stress on synaptic functions in the basolateral amygdala (BLA), one of the key nodes for regulation of social behavior, in mice. [Methods] Male C57BL6/J mice were isolated during postweaning adolescent period and evaluated emotional behaviors and synaptic functions in BLA by whole-cell patch-clamp method after maturation. [Results and conclusion] Isolated mice significantly decreased social preference and increased impulsive aggression. On the other hand, there were no significant difference in anxiety-like behaviors in the open-field and the elevated plus maze tests between ctrl and isolated mice. Furthermore, isolated mice showed lower AMPA/NMDA current ratio in BLA synapse compared with ctrl mice. These results suggested that social isolation rearing in adolescence disrupted the development of excitatory synaptic function in BLA and impaired social function in mice.

Stress is caused by various adverse environments, and often causes emotional changes. Using social defeat stress (SDS) in mice, we previously reported that repeated SDS activates microglia in the medial prefrontal cortex (mPFC), and then leads to social avoidance. Recently, we found that microglial activation in the mPFC occurs more rapidly and strongly with repetition of SDS. This finding led us to speculate that repetition of SDS induced persistent epigenomic changes of microglia in the mPFC. However, due to the limited sensitivity of chromatin immune precipitation sequencing (ChIP-seq), a brain region- and cell type-specific epigenomic analysis has been challenging. Here we optimized the protocol of ChIP-seq for epigenomic analyses of mPFC microglia. This protocol allows us to detect enrichment of active histone marks near microglia-specific genes in mPFC microglia. Our preliminary findings reveal that whole-genome epigenomic profiles of microglia reflected the experience of single SDS or repeated SDS. Interestingly, these epigenomic profiles in mPFC microglia were different from the profiles in the nucleus accumbens, indicating brain-region-specific epigenomic changes. We are currently investigating transcription factors related to regulation of mPFC microglial epigenomic changes associated with emotional changes.

Animal studies using various stress models have shown that excessive environmental stress induces depression-like behaviors with concomitant dendritic atrophy of neurons especially in the medial prefrontal cortex (mPFC) and the hippocampus. Recent progresses have revealed that inflammatory responses in the brain and periphery are critical inducers of dendritic atrophy of the neurons. However, how brain inflammation leads to altered morphology and thereby dysfunction of mPFC neurons remains unknown. Here we aimed to examine the cell-to-cell interaction of microglia and neurons in the mPFC using three-dimensional electron microscopy. We subjected male C57BL/6 mice to either single or repeated social defeat stress, and analyzed the brains from those stressed mice or from control mice which did not receive defeat stress by serial block face-scanning electron microscopy. We found that social defeat stress increased microglia-neuron interfaces that are formed between microglial processes and synaptic structures, predominantly presynaptic axon terminals in the mPFC. Our study will pave the way for a better understanding of molecular and cellular mechanisms of brain inflammation for stress-related mental disorders.

Stress-triggered sympathetic activation increases plasma catecholamine levels, resulting in elevations in blood pressure and heart rate. In addition, facilitated secretion of adrenaline and pancreatic glucagon associated with sympathetic activation increase blood glucose level. We previously reported that corticotropin-releasing factor and acute restraint stress elevates plasma catecholamine levels and that prostanoids in the brain, especially in the paraventricular hypothalamic nucleus (PVN), mediates these responses. Therefore, there is a high possibility that brain prostanoids are involved with stress-related changes in glucose dynamics. In this study, we examined effects of glucose infusion on plasma catecholamine levels and brain prostanoid levels under restraint stress in rats. Glucose solution infused intravenously and intracerebroventricularly effectively suppressed stress-induced elevation of plasma adrenaline level. In addition, we found that stress exposure increased thromboxane B₂ in PVN dialysates, and that the increase in thromboxane B₂ level was suppressed by glucose infusion. Furthermore, glucose infusion improved stress-triggered cardiac dysfunction. Our results suggest that glucose infusion can ameliorate stress-induced sympathetic activation and cardiac dysfunction, and that prostanoids in the PVN are involved with this mechanism.

Postmortem studies have shown that depressed suicide patients have dysfunctions of serotonergic (5-HTergic) systems in the prefrontal cortex. These 5-HTergic dysfunctions are thought to result from multiple factors including adverse environmental experiences. In this study, to investigate mechanisms by which stressful events cause 5-HTergic dysfunctions, we analyzed function, gene expression and epigenetic modification in 5-HTergic systems in mice exposed to repeated social defeat stress. Extracellular 5-HT levels under basal conditions were increased in the prefrontal cortex of stressed mice. Furthermore, 5-HT uptake and 5-HT transporter expression were decreased in synaptosomes derived from the prefrontal cortex of stressed mice. DNA methylation levels were increased at the CpG island of Slc6a4, a gene encoding 5-HT transporter, in the prefrontal cortex-projecting 5-HTergic neurons of stressed mice. The 5-HT uptake in the prefrontal cortex was correlated with time spent in the open arms in the elevated plus maze test, but not social avoidance or sucrose preference. These findings suggest that stressful events decrease 5-HT reuptake in the prefrontal cortex through decreased expression of 5-HT transporters by DNA methylation at CpG island of Slc6a4.

AMP activated protein kinase (AMPK), metabolic regulating emzyme, distributes whole of body including brain. Although physical exercise ameliorates depressive-like behaviors via AMPK activation, the underlying mechanism is remains unclear. Therefore, we investigated whether an AMPK activator AICAR has antidepressant effect in olfactory bulbectomized (OBX) mice.

OBX mice were administered AICAR for 14 days and then conducted tail-suspension test (TST). Hippocampal proteins were assessed by western blotting and neurogenesis was measured by immunohistochemical method.

AICAR subchronic treatment for OBX mice decreased immobility time in TST. Phosphoryled AMPK, protein kinase C (PKC) ζ, nuclear factor-kappa B (NF-κB), cAMP response element-binding protein (CREB) and the expression level of brain derived neurothrophic factor (BDNF) in OBX mice were elevated by AICAR. Hippocampal neurogenesis in OBX treated with AICAR promoted. Some of the effect of AICAR were attenuated by co-administration of PKCζ inhibitor. Activated AMPK was detected in mature and immature neuron and microglia.

In this study, we showed that AMPK activation may exert antidepressant effect via PKCζ/NF-κB/BDNF/CREB signaling pathways, suggesting that AMPK activator may become a therapeutic target of new anti-depressant.

Chronic kidney disease (CKD) is a risk factor for cerebrovascular diseases, and patients with CKD are susceptible to ischemic and hemorrhagic stroke. Many CKD patients complain of anxiety and other psychiatric symptoms; however, involvement in the mechanism of brain disfunction associated with decreased renal function is unknown. In this study, we analyzed psychiatric symptoms and the changes in the brain of 5/6-nephrectomized (CKD) mice, focusing on redox signaling pathways. In the dark/light box test and the elevated plus maze test, anxiety level of CKD mice was higher than sham mice. Compared with sham mice, sociability was lower in CKD mice using sociability test. In addition, the expression levels of Iba1, a marker of microglia, and GFAP, a marker of astrocytes, were increased in the brain of CKD mice. We also found the increase in the protein expression levels of iNOS and eNOS, but not nNOS, in the brain of CKD mice. Notably, in CKD mice, eNOS dimer to monomer ratio was reduced compared with sham mice, indicating the increase in reactive oxygen species (ROS) production. Furthermore, tyrosinated proteins as well as glutathionylated proteins were increased in the brain of CKD mice. These results indicate that disruption of redox signaling in the brain following decreased renal function causes psychiatric symptoms in mice.

[Background] In pathogenesis of depression, BDNF in the brain play an important role. We found that Shati/Nat8l mRNA is increased in striatum of depression model mice exposed repeated social defeat stress (R-SDS). It is reported that BDNF mRNA levels are increased in any brain regions of Shati/Nat8l KO mice. In this study, we revealed the roles of striatal Shati/Nat8l in depression and mechanism of regulation in BDNF by Shati/Nat8l. 

[Methods] C57BL/6J mice were exposed R-SDS using ICR mice, and the acetylated histone levels of BDNF was measured using chromatin immunoprecipitation. We generated striatal Shati/Nat8l conditional knockdown mice (Shati cKD) using Cre-loxP system. Then we assessed depression-like behaviors in Shati cKD with behavioral experiment after R-SDS, and investigated the effect of Shati/Nat8l to the acetylation of histone.

[results] BDNF mRNA levels in the striatum of mice exposed R-SDS is increased, and acetylated histone levels of BDNF is also increased. In the behavioral experiment, Shati cKD showed the resilience for social defeat stress. BDNF mRNA and acetylated histone levels of BDNF in the striatum is suppressed by knockdown of Shati/Nat8l.

[conclusions] Shati/Nat8l in the striatum play an important role in depression. BDNF in the striatum might regulated resilience for social defeat stress mediated histone acetylation by Shati/Nat8l. Our study suggested the new pathways induce depression-like behaviors, and Shati/Nat8l in the striatum might be a new target for medical tools for depression.

We have recently demonstrated that infusion of eicosapentaenoic acid-derived resolvin E1 (RvE1) into the medial prefrontal cortex (mPFC) exerts antidepressant effects via brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) release and subsequent activation of mechanistic target of rapamycin complex 1 (mTORC1) in a murine lipopolysaccharide (LPS)-induced depression model. In the present study, we examined the roles of BDNF/VEGF release and mTORC1 activation within the mPFC in the antidepressant actions of intranasal (i.n.) administration of RvE1 in LPS-induced depression model mice using the tail suspension and forced swim tests. The results demonstrate that the antidepressant effects of i.n. RvE1 are completely blocked by intraperitoneal injection of an AMPA receptor antagonist NBQX or an L-type voltage-dependent Ca²⁺ channel (L-VDCC) blocker verapamil. We also demonstrate that the antidepressant effects of i.n. RvE1 are blocked by intra-mPFC infusion of a BDNF neutralizing antibody (nAb), a VEGF nAb or an mTORC1 inhibitor rapamycin. Together, the current results suggest that the antidepressant actions of i.n. RvE1 are mediated by BDNF/VEGF release which is probably caused by activation of AMPA receptors and L-VDCCs, and subsequent activation of mTORC1 in the mPFC.

Effects of long-period sterile housing on brain functions were assessed using aged germ-free mice at 72 to 75 weeks of age.

We conducted a novel object recognition test as the behavioral test in a germfree environment, measurement of monoamine concentration in the brain in the biochemical test, and Iba1 immunostaining as an indicator of microglial activation in the histopathological examination.

In the novel object recognition test, no difference from the SPF mice of the same age was seen in the aged germ-free mice. However, increased noradrenaline content in the frontal cortex and decreased Iba1-positive cells were noted in the aged germ-free mice.

Recent studies have reported that glutamate receptors (GluR) left-right asymmetry in the rodent brain affects spatial cognition and working memory. However, The relationship between brain left-right asymmetry and individuality has not been studied. Therefore, in this study, we carried out an individuality judgment based on the anxiety-resistant behavior and the anxiety-non-resistant behavior for acrophobia using SD rats, and the animal according to those individuality determined learning ability. We analyzed glutamate receptors in the rat brain hippocampus and hypothalamus. In an individuality judgment, we carried out Improved Elevated Beam Walking Test that increased at height for 3 minutes. We defined the short stay type (S type) and the long stay type (L type) from the residence time on the open bridge. We carried out a two-image discrimination task using a touch screen recognition-learning device for a rat according to the individuality. The hippocampus and hypothalamus were disected from the rat brain, and RT-qPCR analysis was performed using a specific primer of the GluR subtype. The expression level of NR1 mRNA was significantly increased in the right compared to the left in hippocampus. Western blotting could also reproduce the trend. Therefore, it may be inferred that the left-right difference of the NR1 receptor in the hippocampus has an influence on the acrophobia resistance.

The previous study demonstrated that hot water extract of golden oyster mushrooms (Aminothioneine®) and ergothioneine, which is a hydrophilic antioxidant and contained at high levels in golden oyster mushrooms, increased neuronal differentiation in the hippocampal dentate gyrus of mice and have antidepressant-like effects in mice. Here, we investigated the effect of Aminothioneine on BDNF expression in primary cultures of cortical cells and learning and memory in mice. In primary cultures of rat cortical cells, Aminothioneine, but not ergothioneine, significantly increased expression levels of BDNF mRNA. Aminothioneine also increased the levels of BDNF expression in the hippocampus of mice. Aminothioneine significantly increased freezing behavior in the test session of the contextual fear conditioning test. In the test session of the novel object location recognition test, Aminothioneine-administered mice spent more time exploring the object at the novel location. Taken together, Aminothioneine increased expression levels of BDNF in cultured neurons and hippocampus of mice. Also, Aminothioneine enhances learning and memory in mice. Because it has been reported that higher expression levels of BDNF are associated with slower cognitive decline during aging, Aminothioneine could act as a memory stabilizer in an aging brain.

Anthocyanins possess high antioxidant activity and are the major group of polyphenols in blackcurrant, a regional specialty in Aomori prefecture. In this study, we investigated the effect of black currant extracts on cognitive and emotional abnormalities in the senescence-accelerated mouse prone 8 (SAMP8). Four month-old SAMP8 mice were fed a basal diet supplemented with 3% blackcurrant extracts for 2 months, and then behavioral experiments were conducted. In the novel object recognition test, treatment with blackcurrant extracts improved the memory impairment in SAMP8 mice. In addition, reduced anxiety-like behavior in SAMP8 mice was reversed by the extracts in the elevated plus maze test. These results suggest that supplementation of blackcurrant extracts has the potential to improve cognitive and emotional abnormalities in aging as well as age-related neurodegenerative diseases such as Alzheimer's disease.

Food-derived antioxidant ergothioneine (ERGO) is not synthesized in mammals, but ingested from daily diet. After oral administration, ERGO is efficiently absorbed in gastrointestinal tract and distributed to the brain parenchyma via transporter SLC22A4, implying its fundamental role in the brain. Oral administration of ERGO actually enhances learning and memory in mice at least in part through activation of tropomyosin receptor kinases B (TrkB), a receptor for neurotrophins. Therefore, we hypothesized that amount of phosphorylated-TrkB (p-TrkB), the activated form in extracellular vesicles (EVs) in blood may reflect the ERGO-induced enhancement of cognitive function, since EVs may contain brain-derived exosomes and might be useful as a liquid biopsy for verifying the effect on brain function even in humans. We first evaluated expression of p-TrkB by western blot in EVs which were isolated from human serum by ultracentrifugation method. There was a significant correlation between blood ERGO concentration and expression of p-TrkB in EVs. We also confirmed expression of SNAP25, a neuron-derived protein in the isolated EVs. These results suggest that expression of p-TrkB in EVs in circulation is associated with ERGO exposure and might be a possible biomarker useful for assessment of ERGO-induced beneficial effect in the human brain.

Sleep spindles are distinctive EEG waves during NREM sleep, with the frequency in the range 9-15 Hz. Altought the thalamocortical (TC) system is considered as a main source of sleep spindles, several studies indicate that also cortical interneurons may play pivotal roles in generating sleep spindles.

Here, we hypothesized that a certain firing pattern in cortical interneurons is essential for sleep spindles generation, and developed a minimum computational model of an inhibitory cortical neuron with five channels and a pump, which recapitulates the firing pattern of cortical neurons during sleep spindles. Comprehensive bifurcation and detailed mathematical analyses predicted that one channel family play a role in generating the electrophysiological characteristics of sleep spindles. Then, we generated transgenic mice and analyzed sleep spindles from EEG data. As a result, a part of transgenic mice showed decreased sleep spindles episode and events. Combining these two approaches, we suggest a novel mechanism of sleep spindles generation, which may become a potential target for a treatment or a biomarker of certain diseases related to sleep spindles, like schizophrenia or absence seizure.

Glutathione (GSH) plays a key role in antioxidant system especially in the brain. Decreased GSH levels are known to be associated with neurodegenerative diseases. We previously reported that an inhibitor of miR-96-5p, which regulates neuronal GSH synthesis through regulating the protein levels of excitatory amino acid carrier 1 (EAAC1), contributed the neuroprotection against oxidative stress. In this study, we focused on GTRAP3-18, the negative regulator of EAAC1, as a new target of miR-96-5p.

Since GTRAP3-18 was predicted to be indirectly regulated by miR-96-5p, we have tried to identify the GTRAP3-18 regulator which mediate the miR-96-5p regulation using mass spectrometry analysis. And then, we have tested whether these candidate proteins could directly regulate the expression of GTRAP3-18.

The result shows that GTRAP3-18 is up-regulated by miR-96-5p because one of the RNA-binding proteins (RBPs), which is directly regulated by miR-96-5p, negatively regulates the GTRAP3-18. Furthermore, the knockdown of this RBP expression by the specific siRNA transfection increased the expression of GTRAP3-18.

These findings indicate that the regulation of GTRAP3-18 by miR-96-5p is mediated by the RBP.

Synucleinopathies such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB) are neurodegenerative disorders featured by the abnormal accumulation of α-synuclein protein (α-syn). Recent studies showed exogenous pre-formed fibrils (PFFs) of recombinant α-syn induced endogenous α-syn aggregates in human SH-SY5Y cells. In this study, we focused on nicotinic acetylcholine receptor (nAChR) as the therapeutic targets for synucleinopathies through α-syn clearance. PFFs were generated from α-syn by agitation followed by sonication. SH-SY5Y cells overexpressing α-syn gene were treated with PFFs for 24 h. After the incubation for 48 h, immunocytochemical analysis revealed the localization of α-syn. PFFs induced the aggregation of α-syn, which were detected with anti-phosphorylated-α-syn antibody and thioflavin-T. These data indicated that intracellular aggregation of α-syn were Lewy body-like aggregates. In addition, we examined galantamine effect, because some reports showed nAChR signaling increased autophagic activity. Galantamine were treated for 48 h after preincubation with PFFs, and then α-syn aggregation was decreased. These observations suggest that nAChR may be a novel target for synucleinopathies through α-syn clearance by autophagic activation.

Chaperone-mediated autophagy (CMA) mediates the selective lysosomal degradation of cytosolic proteins. CMA activity is regulated by the expression and complex formation of LAMP2A on lysosomal membranes. We have revealed that CMA impairment is commonly observed in cells expressing several types of mutant proteins causing spinocerebellar ataxia (SCA), characterized by the progressive cerebellar ataxia. Therefore, we assumed that the decrease in CMA activity is related to the pathogenesis of SCA. In the present study, we investigated how CMA impairment by LAMP2A knockdown affects motor function of mice. LAMP2A knockdown was conducted by the cerebellar injection of adeno-associated viral vectors, which express GFP and miRNA against LAMP2A in a neuron specific manner, to 4-week old ICR mice. Motor performances of mice was evaluated by beam walking test. LAMP2A knockdown in cerebellar neurons triggered the progressive motor dysfunction. Immunostaining of cerebellar sections revealed that GFP was mainly expressed in interneurons of molecular layer and granule cells. In addition, decrease in Purkinje cells and shrinkage of molecular layer were observed around GFP-positive neurons. These results suggest that CMA impairment in cerebellar neurons causes ataxic phenotype and degeneration of Purkinje cells.

Parkinson's Disease (PD) is a common neurodegenerative disease. Although more than 20 causal genes for PD have been identified from patients with familial PD, the majority of PD cases are sporadic and the detailed onset mechanism remains unclear. Our previous genetic analysis revealed that the copy number loss of Midnolin (MIDN) is found in 10.5% of patients with sporadic PD in a Yamagata population cohort. Furthermore, we showed that MIDN regulates neurite outgrowth and expression of Parkin E3 ubiquitin ligase in PC12 cells. Here, we attempted to replicate the genetic association between MIDN and PD in a large British population cohort. We examined copy number variations and single nucleotide polymorphisms of the MIDN gene in 2,860 controls and 2,168 PD patients. We found significant copy number loss in the MIDN gene in 6.55% of patients with PD (odds ratio = 4.35). In addition, when a deletion spanning more than 50,000 bp was defined as the copy number loss, the odds ratio dramatically increased to 22.3. No significant differences for two frequent single nucleotide polymorphisms (rs3746106 and rs3746107) were found. Taken together, we show the strong genetic association of MIDN with PD development in a British population and in a Japanese population, suggesting MIDN is a universal genetic risk factor for PD.

Several epidemiological studies suggest that uric acid exerts a neuroprotective effect in neurodegenerative disease such as Parkinson's disease and Alzheimer's disease (AD). However, the molecular mechanism how uric acid affect the pathology and/or cognitive function in Alzheimer's disease remains unclear. In this study, we developed a combined mouse model by cross-breeding App^NL-G-F knock-in mouse（App-KI）which carries humanized amyloid βprotein (Aβ) sequence with familiar AD-associated mutations, and uricase knockout mouse (Uox-KO) which shows increased level of uric acid. To prevent renal failure caused by elevated uricnary excretion of uric acid, App-KI-Uox-KO mice were treated with allopurinol by dietary administration. We performed immunohistochemical staining for Aβwith anti-Aβ, astrocytes with anti-GFAP, and micloglia with anti-Iba1 using brain sections from 8 month-old mice. Aβ accumulation was increased in App-KI-UOX-KO mice in comparison with App-KI mouse. However, App-KI-Uox-KO mice displayed reduced astrocytosis and microgliosis in the cortex. Further studies are required to determine whether the glial changes are the cause or consequence of increased Aβ accumulation under higher uric acid level.

Oxidative stress has been implicated in the progression of neurodegenerative disorders, including Parkinson's disease. We have previously demonstrated that noradrenaline increases the level of intracellular glutathione (GSH) in astrocytes via β₃-adrenoceptor stimulation and protects co-cultured neurons from oxidative stress-induced death by increasing the supply of GSH from astrocytes. In addition, we have reported that the intraperitoneal administration of SR58611A, a β₃-adrenoceptor agonist, increases the level of GSH in ventral midbrain. In the present study, we investigated the effects of SR58611A on the degeneration of dopaminergic neurons induced by 6-hydroxydopamine (6-OHDA) in C57BL/6 mice. Mice were injected with 6-OHDA into the right striatum, and the degeneration of dopaminergic neurons was evaluated by tyrosine hydroxylase (TH) immunohistochemistry. At 14 days after the intrastriatal injection of 6-OHDA, the number of TH-positive cells was decreased in substantia nigra, and this decrease was attenuated by treatment with SR58611A (once a day 5 mg/kg, i.p. for 3 days). These results suggest that SR58611A attenuates 6-OHDA-induced degeneration of dopaminergic neurons by increasing brain GSH levels and that β₃-adrenoceptor agonists may be useful as novel therapeutic agents for the treatment of Parkinson's disease.

Calmodulin-like skin protein (CLSP), a secreted peptide, likely protect neurons from toxicity linked to Alzheimer disease (AD) via the heterotrimeric humanin receptor. We have addressed the issues regarding the clinical relevance of CLSP regulation in the AD pathogenesis and the potential of some CLSP derivatives named CLSP(1-61) and CLSPCOL as anti-AD agents by preclinical tests. By conducting an in vitro AD-related death assay, we have characterized endogenous CLSP interactors. By measuring the levels of CLSP interactors in AD-patient-derived samples, we have next examined whether the dysregulation of CLSP interactors contributes to the AD pathogenesis. Finally, by administering potent CLSP derivatives to AD models, we have tested their potentials as anti-AD therapeutic agents. Some CLSP-binding proteins including apolipoprotein E inhibited the CLSP-mediated neuroprotective effect. Oppositely, adiponectin enhances the CLSP activity and enabled CLSP to remain fully active even in the presence of overwhelming amounts of CLSP inhibitors, by binding to the humanin-homologous region of CLSP. We further show that both the level of adiponectin and the intraneuronal level of SH3BP5, a central intraneuronal signal transducer of the humanin/CLSP effect, were downregulated in AD patients. We designed CLSP(1-61) and CLSPCOL as anti-AD agents that are free from the suppression of CLSP inhibitors and tested their ant-AD efficacy. Adiponectin dominantly determines the CLSP activity. The downregulation of the AD-protective activity of CLSP, caused by the reduction of brain adiponectin, may contribute to the AD pathogenesis. CLSP-derived peptides, CLSP(1-61) and CLSPCOL, may be ideal disease-modifying anti-AD agents.

OBJECTIVE: The incidence of Alzheimer-type dementia has been reported to be higher in patients with diabetes. Recently, excessive glycation (Advanced glycation end-products; AGEs) of neuronal proteins by hyperglycemia is considered as risk factor for Alzheimer-type dementia. The intrauterine hyperglycaemia during pregnancy has various effects on foetal development. Therefore, we examined the effect of excessive glycation in the neuronal cells by the intrauterine hyperglycaemic environment during foetal brain development.

METHODS: We analysed the AGEs and Akt related signalling of neuronal cells of infant of diabetic mother rat (IDMs). In addition, we examined the effects of high glucose culture medium on the rat PC12 cells.

RESULTS: In the brains of IDMs, AGEs were 1.3 times higher than that in controls, and Akt phosphorylation was 0.78 times lower. The AGEs of PC-12 cells cultured in high-glucose medium was 3.3 times higher and Akt phosphorylation was 0.59 times lower than that in cells cultured in low-glucose medium. Furthermore, apoptosis was enhanced in the high glucose medium.

CONCLUSION: We have shown that a hyperglycemia promotes excessive protein glycation in neuronal cells and may be more likely to degenerate of neuronal protein, the possibility of linked to increased Alzheimer's risk in the long-term.

We have shown previously that prostaglandin E2 (PGE2) directly induces neuronal death through activation of the E-prostanoid (EP) 2 receptor in differentiated NSC-34 cells, motor neuron-like cell line. In the present study, to clarify the mechanisms underlying PGE2-induced neurotoxicity, we focused on generation of intracellular reactive oxygen species (ROS) in NSC-34 cells. Dichlorofluorescein fluorescence analysis of PGE2-treated cells showed that intracellular ROS levels increased markedly with time, and that this effect was antagonized by an EP2 antagonist, PF-04418948, but not EP3 antagonist, L-798,106. Although an EP2 agonist, butaprost, mimicked the effect of PGE2, an EP1/EP3 agonist, sulprostone, transiently but significantly decreased the intracellular ROS. MTT reduction assay and lactate dehydrogenase release assay revealed that PGE2- and butaprost-induced cell death were each suppressed by pretreatment with a cell permeable antioxidant, N-acetylcysteine (NAC). Western blot analysis revealed that the active form of caspase-3 was markedly increased in the PGE2- and butaprost-treated cells. These increases in caspase-3 protein expression were suppressed by pretreatment with NAC. Our data have demonstrated that PGE2 is an endogenous inducer of intracellular ROS, and that production of ROS induced by PGE2-EP2 receptor signaling is coupled to the caspase-3 cascade in NSC-34 cells.

15-deoxy-Δ^12,14-prostaglandin J₂ (15d-PGJ₂) is one of factors contributed to the neurotoxicity of amyloid β, a causative protein of Alzheimer's disease. A proteomic approach with biotinylated 15d-PGJ₂ was used to identify its targets in the plasma membrane of rat cortical neurons. Previously, we have identified plasmalemmal targets as biotin-positive spots and classified into three functional proteins: glycolytic enzymes (enolase 1, enolase 2, pyruvate kinase isozymes M1/M2 and glyceraldehyde 3-phosphate dehydrogenase), molecular chaperones (heat shock protein A8 and T-complex protein 1 subunit α), cytoskeletal proteins (Actin β, F-actin-capping protein, tublin β, internexin α and glial fibrillary acidic protein). In the present study, we identified anion channel and adaptor proteins as membrane targets of 15d-PGJ₂. These novel targets are known as mitochondrial or cytosolic proteins, suggesting their ectopic localization. 15d-PGJ₂ possesses opposite functions as a neuroprotectant at low concentrations and a neurotoxicant at high concentrations in the brain. Its nuclear receptor, peroxysome proliferator-activated receptor-γ , contributes to the neuroprotective effect of 15d-PGJ₂, but not to the neurotoxic effect. Its membrane receptor, chemoattractant receptor-homologous molecule expressed on T-helper type 2 cells , is not also involved in the neurotoxicity. Thus, we discussed how 15d-PGJ₂ could inhibit neurite outgrowth and induce neuronal apoptosis via these novel targets.

Aquaporin-4 (AQP4) is the most abundant water channel in the CNS specifically expressed in the astrocytic end-feet around blood vessels and is thought to contribute to water and ion homeostasis in the brain. AQP4 has been suggested to be involved in the pathogenesis of neurodegenerative diseases including Alzheimer's disease (AD), which may be due to the modulation of neuroinflammation or the impairment of interstitial fluid bulk flow system in the central nervous system. In this study, to investigate the implication of AQP4 in AD pathology, we crossed an AD model, 5xFAD with AQP4 knockout mice. We demonstrate that deleting AQP4 from 5xFAD resulted in acceleration of behavioral abnormality, namely an age-dependent reduction of nighttime activity, associated with aggravation of epileptiform neuronal activity as well as convulsions. Importantly, these symptoms occurred independently of accumulation of amyloid plaques or neuroinflammatory responses of glial cells, since in this model, AQP4 deficiency did not affect age-dependent increase in amyloid deposition in parenchyma or neuroinflammation. Our results provide an important perspective for developing new diagnostic methods and treatments for Alzheimer's disease.

Tau is one of the characteristic deposits in Alzheimer's Disease which occurs in neurofibrillary tangles within neurons. Positron-emission tomography (PET) is a nuclear imaging technique, making it possible to detect tau aggregation in AD in vivo, thereby helping to pre-diagnose and trace disease progression. Up to now, many tau tracers have been developed. For example, the first generation tau tracer [¹⁸F]AV-1451 and [¹⁸F]THK-5351 developed by our group displayed strong uptake in patients' brains. Although they were promising, considerable off-target binding has been observed, especially to MAO-B and other non-tau protein deposits. This led to the optimization of binding characteristics and discovery of novel tau tracers. 

More than 200 candidates synthesized by our group were screened through binding assay for tau, amyloid β, monoamine oxidase A and B. Among these compounds, [¹⁸F]THK-5562 was identified with high affinity and selectivity for tau aggregation without off target bindings, followed by further evaluation. As a result, [¹⁸F]THK-5562 displayed excellent in vivo pharmacokinetics characteristics in mice. Besides, [¹⁸F]THK-5562 seemed to be specific for 3R/4R tau in AD by autoradiography and immunohistochemistry.

In conclusion, preclinical validation suggested the potential usefulness of [¹⁸F]THK-5562 in imaging tau aggregation in Alzheimer's disease.

Recently, we reported that centrally administered SIN-1, a donor of nitric oxide (NO), induced frequent urination in rats. In the present study, therefore, we investigated central mechanisms how brain NO induced frequent urination focusing on glutamatergic receptors in urethane-anesthetized (0.8 g/kg, ip) male Wistar rats. A catheter was inserted into the bladder to perform cystometrograms (CMG). CMG was started 2 h after the surgery and 1 h after the start, SIN-1 (250 μg/rat) was intracerebroventricularly (icv) administered. Effects of icv pretreated carboxy-PTIO (PTIO, NO scavenger, 750 μg/rat), MK-801 [N-methyl-D-aspartate (NMDA) receptor antagonist, 10 or 30 nmol/rat] or DNQX (AMPA receptor antagonist, 3 nmol/rat) on the SIN-1-induced responses were also investigated. SIN-1 dose-dependently shortened intercontraction intervals (ICI). The SIN-1-induced ICI shortening was significantly attenuated by central pretreatment with PTIO or MK-801, respectively. On the other hand, DNQX showed no significant effect on the SIN-1-induced ICI shortening. These results suggest that brain nitrergic pathway can directly regulate the micturition reflex via brain NMDA receptors in rats.

Peripheral hydrogen sulfide (H₂S) is reported as an endogenous relaxation factor in the bladder, while in the central nervous system, roles of H₂S in regulation of micturition is unclear. In this study, therefore, we examined effects of centrally administered GYY4137 (GYY, H₂S donor) or AOAA (H₂S synthesis inhibitor), on the micturition reflex in urethane-anesthetized (0.8 g/kg, ip) male Wistar rats. A catheter was inserted into the bladder to perform cystometrograms (CMG). CMG was started 2 h after the surgery and 1 h after the start, GYY (3 or 10 nmol/rat) or AOAA (30 or 100 μg/rat) was intracerebroventricularly (icv) administered. Effects of icv pretreated SR95531 (SR, GABA_A antagonist, 0.1 nmol/rat) or SCH50911 (SCH, GABA_B antagonist, 0.1 nmol/rat) on the GYY-induced responses were also examined. GYY dose-dependently prolonged intercontraction intervals (ICI), while AOAA dose-dependently shortened ICI. The AOAA-induced ICI shortening was reversed in the presence of GYY. Pretreatment with SR or SCH significantly attenuated the GYY-induced ICI prolongation. These results suggest that brain H₂S can inhibit the micturition reflex via brain GABA_A and GABA_B receptors in rats.

Background：It has been reported that the incidence of cardiovascular diseases were increased after acute kidney injury (AKI). On the other hand, there are few reports that evaluated vascular endothelial function underlying cardiovascular risk after AKI. We have previously confirmed that vascular endothelial function declines after AKI. This study, we examined the relationship between vascular endothelial dysfunction in AKI and indoxyl sulfate (IS) and NO bioavailability.

Methods： Ischemic AKI (IAKI) model was prepared by renal ischemia reperfusion (IR). One, 7 and 28 day after IR, blood and urine were collected, and IS concentration were measured. NO bioavailability was evaluated by acute treatment with arginine in organ bath.

Results：One day after IR, the IS clearance was decreased, and it recovered to the same extent as sham with the recovery of renal function. The attenuation of vascular endothelial function observed in IR was significantly improved by the arginine acute treatment and the combined treatment with arginine and arginase inhibitor.

Conclusion：These results suggest that a temporary increase in uremic toxin associated with decreased renal function partially contributes to vascular endothelial dysfunction in AKI. Furthermore, the decline of utilization of arginine may be involved in the above phenomena.

Sepsis-induced acute kidney injury (AKI) is frequently observed in the intensive care unit. We previously revealed that yohimbine, α2-adrenoceptor antagonist, has protective effect against renal ischemia/reperfusion injury-induced AKI in rats. This study aimed to investigate the renoprotective effect of yohimbine against lipopolysaccharide (LPS) - induced AKI in rats. Male Sprague Dawley rats were randomly divided into following groups: Sham-operated group, LPS (10 mg/kg, i.p.), LPS + yohimbine (0.1 or 0.5 mg/kg, i.p.). Kidney functional parameters of blood urea nitrogen (BUN), plasma creatinine (Pcr) were aggravated in LPS group. Administration of LPS decreased mean arterial blood pressure. In addition, kidney injury molecule-1, iNOS and various cytokine expression such as TNF-α, MCP-1 and IL-6 were increased by LPS administration. The treatment with yohimbine clearly ameliorated damaged kidney function and blood pressure due to LPS. Moreover, yohimbine suppressed cytokine mRNA and iNOS expression enhanced by LPS. However, anti-inflammatory cytokine IL-10 mRNA levels were augmented by yohimbine. NF-kB nuclear translocation in kidney was observed 1 hr after injection of LPS in rats. Yohimbine blocked nuclear entry of NF-kB. In addition, phosphorylation of ERK and CREB were enhanced with yohimbine. These results suggest that yohimbine can prevent LPS-induced sepsis associated with kidney injury by suppressing the inflammatory cytokine expression and enhancement of IL-10 expression via ERK/CREB phosphorylation.

Chronic kidney disease (CKD) is characterized by progressive and chronical kidney dysfunction. Although CKD can be categorized into various types based on pathogenesis, adriamycin (ADR) -induced nephrotic syndrome is considered to be a classical rat model of CKD. By administration of ADR, glomerular filtration barrier damage and subsequent massive proteinuria are induced. In this study, we tried to prepare an ADR-induced nephropathy model in rats (Experiment 1) and to validate the usefulness of the model by administration of prednisolone (Experiment 2). In experiment 1, ADR was administered intravenously to Crlj:WI rats at 3, 5 or 10 mg/kg. Only the 3 mg/kg group, 2 mg/kg of ADR was additionally administered 15 days after the first ADR administration. 24hr-urine and blood were collected once weekly for 6 weeks for urinalysis and blood chemistry-analysis. Results indicated that ADR at 5 mg/kg was found to be suitable for induction of the nephropathy model rats. Therefore, in experiment 2, ADR was administered at 5 mg/kg, and prednisolone was administered orally at 1 or 5 mg/kg once a day for 35 days. Prednisolone was observed to be effective on dysfunction of the kidney by urinalysis and blood chemistry-analysis, indicating that ADR-induced nephropathy model is useful in evaluation of developing therapeutic drugs under the present experimental condition.

Lysophosphatidic acid (LPA) is a bioactive lysophospholipids that regulates multiple biological functions. Several studies showed an increased urinary LPA level in chronic kidney disease (CKD) patients and animal models, suggesting that the local expression levels of LPA and LPA receptor subtypes are important for the development of CKD. However, the role of LPA in the transition of acute kidney injury (AKI) to CKD remains unknown. Renal ischemia/reperfusion (IR) can induce AKI, which often progresses to CKD. To study the role of LPA on the transition of AKI to CKD, we used a murine unilateral renal IR injury (uIRI) model without contralateral nephrectomy. On 14 days after IR, interstitial fibrosis and renal atrophy were observed. Real-time PCR analysis demonstrated significant upregulation in the expression of LPA type1 receptor subtype (LPA₁) and LPA₂ and downregulation of LPA₃. We also examined the involvement of LPA₁ in the development of interstitial fibrosis and renal atrophy. Pharmacological inhibition of LPA₁ with Ki16425 did not affect interstitial fibrosis and atrophy by IR. These results suggest that further investigation including the involvement of other LPA receptor subtypes is necessary.

We have confirmed that hydrogen sulfide (H₂S) is a possible relaxation factor in the rat bladder, and H₂S-induced bladder relaxation is impaired in 18-week-old (18W) spontaneously hypertensive rats (SHRs), which show bladder dysfunctions. We compared effects of NaHS and GYY4137 (H₂S donors) on bladder contractility and micturition reflex, and H₂S contents and expression of enzymes related to H₂S biosynthesis (CBS, MPST and CAT) in the bladder between 12W and 18W male SHRs. Effects of NaHS (1×10^-8 to 3×10^-4 M) were evaluated on carbachol (10^-5 M)-induced pre-contracted bladder strips. Under urethane-anesthesia, effects of intravesically instilled GYY4137 (10^-8 to 10^-6 M) on the rat micturition reflex were examined. The H₂S contents and expression of CBS, MPST and CAT were measured by methylene blue method and western botting. Compared to 12W SHRs, NaHS-induced maximal relaxation of bladder strips was significantly decreased, GYY4137-induced intercontraction intervals prolongation was attenuated, the bladder H₂S content and expression level of CBS, MPST and CAT in the bladder dome was higher in 18W SHRs. These results indicate that H₂S-induced bladder relaxation in SHRs is impaired time-dependently, suggesting that early intervention in SHRs with H₂S donors may prevent the development of hypertension-mediated bladder dysfunctions.

Unilateral ureteral obstruction (UUO) is a well-established model for the study of interstitial fibrosis in the kidney. In this study, we investigated effects of two antioxidants, N-acetyl cysteine (NAC) and tempol, on UUO-induced renal interstitial fibrosis in mice. Fibrotic area of Masson trichrome-stained section significantly increased by UUO. This change was inhibited by the treatment of NAC, but not by tempol. Tempol administration increased the amount of hydrogen peroxide in the kidney. Catalase activity was not different between NAC and tempol. Glutathion peroxidase activity was inhibited by UUO, and this effect was diminished by NAC. Collectively, these results suggest that NAC may increase the glutathion peroxidase activity, ameliorating the renal interstitial fibrosis induced by UUO.

The E3 ubiquitin ligase, parkin, regulates protein stability by promoting polyubiquitination and degradation. Although parkin is known to play an essential role in cardiac function after myocardial infarction (MI) by promoting degradation of damaged mitochondria via autophagy, its physiological significance in the other cardiac diseases such as dilated cardiomyopathy (DCM) remains unclear. Here, we found that the expression level of parkin was downregulated in cardiomyocytes from DCM mouse hearts. Conversely, the interaction between parkin and phospholamban (PLN), a potent inhibitor of sarco(endo)plasmic reticulum Ca²⁺-ATPase, was increased in cardiomyocytes from DCM mouse hearts in the age-dependent manner. It was also shown that the interaction between PLN and PTEN-induced putative kinase 1 (PINK1), which has been shown to cooperate with parkin to promote polyubiquitination and degradation of mitochondrial proteins to regulate mitochondrial dynamics, was enhanced. These data indicate that the enhanced interaction of PLN with PINK1 as well as with parkin leads to the increased degradation of PLN in failing hearts. The degradation of PLN by parkin and PINK1 might be one of the compensating mechanism to maintain the cardiac function.

In the failing heart following myocardial infarction (MI), mitochondrial dysfunction induces myocardial apoptosis and cardiac pump failure. Heat shock protein (HSP) A9 contributes to mitochondrial homeostasis. However, pathophysiological roles of HSPA9 during the development of heart failure following MI remain unclear. In this study, we assessed changes in HSPA9 in the left ventricle (LV) during the development of heart failure following MI. MI was induced by the left coronary artery ligation (CAL). At 8th week after CAL (8W-CAL), cardiac output index and mitochondrial activity were reduced. HSPA9 in the mitochondria-enriched fraction of the 8W-CAL animals were determined. In the cytosolic and nuclei-enriched fractions, HSPA9 of the 8W-CAL animals were increased. In the co-immunoprecipitation of the LV, the apoptotic signaling protein p53 in the HSPA9 immunocomplex of the 8W-CAL animal was increased. These findings suggest that HSPA9 accumulation in the mitochondria contributes to keep the LV function after MI. In contrast, the accumulation of the complex of HSPA9 and p53 in cytosolic and nucleic fractions is a possible mechanism to induce apoptosis of cardiomyocytes during the development of heart failure following MI.

Nowadays, exercise is regarded as one of therapies for heart failure (HF). However, the effects of exercise on patients with dilated cardiomyopathy (DCM) have not been established. A knock-in mouse model of human inherited DCM, TNNT2 ΔK210, shows similar characteristics to DCM patients. We have recently found that voluntary exercise significantly improves cardiac function in DCM mice. In this study, we focus on differences in the effects of voluntary exercise due to sexual specificity in the DCM model mice. 

Homozygous ΔK210 (DCM) mice showed enlarged heart and frequent sudden death with t1/2 of ～70 days. Male and female DCM mice started running using wheels at 1 month of age. The non-exercise controls of both genders were housed without wheels. After running for a month and two months, mice were investigated with echocardiography. After sacrifice, weights of body, heart, lung, lower extremity muscles were measured. Gene expressions of HF- and arrhythmia-related genes in myocardium were quantified by qPCR analysis. 

At 2 months of age, the ejection fraction (EF) were significantly improved in male exercise group compared with male control group (exercise group:36.9±10.0 (n=5)%, control group:21.2±7.9% (n=7)) However, in females, the EF of both groups did not differ significantly (exercise group:27.5±10.1% (n=7), control group:23.1±9.6% (n=5). We will also show the results of gene expression analysis and discuss reasons for the difference in effects of voluntary exercise.

The cardiosphere-derived cell (CDC) is one of the candidate cells used for cardiac regenerative therapy. Cardiospheres are mixture of CDCs including c-Kit⁺ cells, Sca-1⁺ cells, and other types of cardiac progenitor cells. In this study, we compared effects of transplantation of isolated Sca-1⁺ cells and c-Kit⁺ cells with that of the crude CDCs (CrCDCs). We found that the transplantation of these 3 types of cells resulted in a preservation of the cardiac pump function and mitochondrial respiration. However, mitochondrial function in the c-Kit⁺ cell-transplanted group was lower than that in the other 2 experimental groups. Furthermore, we found that activation levels of intracellular signaling proteins after cell transplantation may have been development on the ability of secretion of several growth factors, such as IGF-1 and TGF-b, by these transplanted cell types. Our findings suggest the possibility that CrCDC and Sca-1⁺ cells rather than c-Kit⁺ cells are preferable for the therapy to preserve cardiac function and energy metabolism after myocardial infarction.

It is known that Bcl-2 associated athanogene (BAG) 3 is strongly expressed in cardiac muscle as well as skeletal muscle. BAG3 can directly bind to Bcl-2 as well as heat shock protein (HSP) as a co-chaperone. Recent study showed that myofibrillar degeneration, disruption of Z-disk architecture and apoptotic cell death were observed in BAG3 knockout mouse. Thus, BAG3 may play a protective role in the muscles. We examined BAG3 protein levels in alpha-B crystallin (CryAB) R120G transgenic (TG) mouse, myofibrillar myopathy (MFM) model. A marked increase in BAG3 was observed in MFM hearts. Little is known, however, detail roles of the increased BAG3 in cardiac muscle. In order to understand functional role of increased cardiac BAG3 in MFM hearts, CryAB R120G TG mice were crossbred with TG mice overexpressing BAG3 to generate CryAB R120G/BAG3 double TG mice. Decrease in fractional shortening and induction of cardiac ANP as well as increase in heart weight/body weight ratio were seen in CryAB R120G TG mice. Moreover, deterioration in cardiac function as well as enhanced cardiac hypertrophy were observed in the CryAB R120G/BAG3 double TG mice. Thus, cardiac BAG3 overexpression may be insufficient for prevention of cardiac disease in CryAB R120G TG mice.