神経化学教育口演

神経化学教育口演 Diseases and Cures

2G2-01 Effect of a novel cognitive enhancer ST101 on decreased CaMKII activity in schizophrenia model rats. Yabuki Yasushi，Fukunaga Kohji Dept. Pharmacol., Tohoku Univ. Grad. Sch. Pharm. Sci. [Background]Development of cognitive enhancer is essential to improve quality of life for schizophrenia patients. We used neonatal ventral hippocampus（NVH）-lesioned rats as schizophrenia model animal, in which risperidone improves sensory motor gating deficits. However risperidone, a typical anti-psychotics fails to improve cognition assessed by novel object recognition test. Notably, cognitive impairment of NVH-lesioned rats is associated with the decreased CaMKII activity in the medial prefrontal cortex（mPFC）and hippocampus（Yabuki et al., Neuroscience 2013；234：103-115）. Therefore, we here investigated the effect of a novel cognitive enhancer ST101（piro[imidazo[1,2-a]pyridine-3,2-indan]-2（3H）-one）on the cognitive impairment of NVH-lesioned rats and its mechanism. [Methods]To prepare NVH-lesioned rats, ibotenic acid was injected into the bilateral ventral hippocampus on the postnatal day（PD）7. After PD 70, animals were administrated with ST101（0.01, 0.1, or 0.5 mg/kg, p.o.）once a day for 2 weeks and were subjected to cognition tests. CaMKII activity was measured using immunohistochemical and western blotting analyses. [Results]The chronic administration of ST101 rescues the decreased CaMKII activity in the mPFC and hippocampus. The cognitive impairment of NVH-lesioned rats was significantly improved by ST101 administration. [Conclusion]NVH-lesioned rats are potential animal model of schizophrenia because the animals show abnormal sensory motor function only after post-pubertal. A novel cognitive enhancer ST101 which is T-type calcium channel stimulator restores cognitive impairment by stimulationg CaMKII activity in the mPFC and hippocampus. Taken together, ST101 is attractive candidate therapeutics for cognitive impairment in schizophrenia patients.		2G2-02 Brain-Active Herbal Metabolites for the Treatment of Alzheimer’s Disease Yang Zhiyou，Kuboyama Tomoharu，Tohda Chihiro Division of Neuromedical Science, Institute of Natural Medicine, University of Toyama Our previous studies have shown that the water extract of a crude drug DR（1, 10 μg/ml）could reverse Aβ（25-35）-induced axonal atrophy in cultured mouse cortical neurons. Administration of DR extract（p.o., for 21-31 days）to Alzheimer’s disease（AD）model, 5×FAD mice, improved deficits of object recognition memory and spatial memory. In this study, we aimed to clarify effects of DR extract on axonal degeneration and AD pathologies in the 5×FAD mice brain. In addition, we tried to identify active compounds detected in the brain, which were distributed constituents or metabolites in DR extract.Oral administration of DR extract（500 mg/kg）for 31 days to 5×FAD（male and female, 6-8 months old）significantly reduced amyloid β plaques in the medial prefrontal cortex, perirhinal cortex and hippocampus. Abnormally swollen degenerated axonal terminals were also significantly decreased in the DR-treated group. To identify DR-derived compounds transferred into the brain, the plasma and cerebral cortex were isolated from DR- or vehicle-treated 5×FAD mice at 0.5 and 5 h after the administration. We had already known about main constituents in DR extract by chemical analyses. Three metabolites in the cortex and thirteen metabolites in the plasma were detected by LTQ-Orbit trap FT-MS/MS analysis. Axonal regeneration activities of those metabolites in the brain are under investigation. To investigate starting point in the signaling mechanism of DR extract, Drug Affinity Responsive Target Stability（DARTS）analysis was performed. By DARTS method using DR extract and mouse cortical lysate, CRMP2 and RKIP were suggested as candidates for direct target proteins of DR constituents. Signaling pathways of DR-derived real active compounds working in the brain are now investigated. Those information may provide a new viewpoint of memory improvement pathway. A name of the crude drug is not open due to patent matters.

2G2-03 Bruton’s tyrosine kinase（BTK）inhibitor has a protective effect on ischemic brain injury. Ito Minako，Shichita Takashi，Yoshimura Akihiko，Morita Rimpei Department of Microbiology and Immunology, Keio University School of Medicine Stroke causes ischemic brain injury, which is a leading cause of neurological disability and death worldwide. The therapeutic time window of t-PA is only 4.5 hours after stroke onset. There is a need for an efficacious therapy that can be administered beyond this time window. Post-ischemic inflammation is a hallmark of ischemic stroke pathology. IL-1β promotes brain tissue injury and is therefore potential targets for therapy after ischemic stroke. Activation of IL-1β is required for caspase-1 activation by formation of inflammasome. We used a transient middle cerebral artery occlusion（MCAO）model（60 min）induced by means of an intraluminal suture. In this study, we demonstrates that The FDA-approved BTK inhibitor ibrutinib（PCI-32765）significantly suppressed infarct volume growth and neurological damage in the brain ischemia/reperfusion model. Ibrutinib suppressed caspase-1 activation and IL-1β secretion by inhibiting the formation of inflammasome.		2G2-04 Immediate or delayed administrations of matrine improve motor dysfunction in spinal cord injured mice Tanabe Norio¹，Kuboyama Tomoharu¹，Kazuma Kohei²，Konno Katsuhiro²，Tohda Chihiro¹ ¹Div. of Neuromedical Science, Inst. of Natural Med., Univ. of Toyama，²Div. of Kampo-Pharmaceutics, Inst. of Natural Med., Univ. of Toyama In spinal cord injury（SCI）, descending neuronal tracts are disrupted, which induce motor dysfunction. Although reconstruction of spinal tracts by axonal growth must be effective for improvement of motor dysfunction, inhibitory factors for axonal extension, such as chondroitin sulfate proteoglycan（CSPG）, increase in the lesion site. We previously found that the water extract of dried roots of Sophora flavescens（SF）promoted axonal extension even on inhibitory CSPG in vitro and improved the axonal density and motor dysfunction in SCI mice. In this study, we aimed to identify active constituents in SF extract and investigate effects of the active constituents in SCI mice. Axonal extension activities of compounds in SF extract were evaluated in cultured cortical neurons（ddY mice, E14）on the CPSG. Four days after the treatment, axonal length was quantified by immunostaining for phosphorylated neurofilament-H. Although axonal growth was inhibited on the CSPG, matrine（10 μM）extended axons even on the CSPG. Consecutive oral administrations of matrine（100 μmol/kg/day, for 30 days）or vehicle solution to SCI mice（ddY, female, 8 weeks old）were started from 1h after the injury. Matrine significantly recovered motor function of hindlimbs. Furthermore, effects of matrine on the motor function in SCI mice by delayed administration were investigated. Matrine（100 μmol/kg/day）was administrated to SCI mice for 154 days from 28 days after the injury. As a result, motor function was significantly recovered by matrine treatment. This study suggests that matrine is one of the active constituents in SF extract and effective for recovery of motor function of SCI mice in chronic phase as well as acute phase. Investigating mechanisms underlying matrine effects is ongoing.

2G2-05 Novel candidate compounds identified by in silico screening activate TrkB and attenuate depressant-like behavior in mice Fukuda Mayu^1,4，Takatori Atsushi^1,2，Nakamura Yohko^1,3，Suganami Akiko⁵，Hoshino Tyuji⁶，Tamura Yutaka⁵，Nakagawara Akira⁷ ¹Div. of Innovative Cancer Therapeutics, Chiba Cancer Ctr. Res. Inst.，²Div. of Cancer Genetics, Chiba Cancer Ctr. Res. Inst.，³Div. of Cancer Prevention and Epidemiology, Chiba Cancer Ctr. Res. Inst.，⁴Grad. Sch. of Med. and Pharmaceutical Sciences, Chiba Univ.，⁵Dept. of Bioinformatics, Grad. Sch. of Med. Chiba Univ.，⁶Grad. Sch. of Pharmaceutical Sciences, Chiba Univ.，⁷Saga Medical Ctr. KOSEIKAN BDNF is a ligand for its receptor TrkB and contributes to neuronal survival, differentiation and synaptic plasticity in central nervous system. Recent reports have indicated that dysfunction of BDNF-TrkB is related with depression. We previously performed in silico screening to find new anti-neuroblastoma（NB）therapeutics targeting BDNF binding domain of TrkB. From 3 million low molecular weight compounds, we finally discovered 7 compounds that induce apoptosis（Nakamura et al., 2014）. In the study, we have also identified 2 distinct compounds（48 and 56）harboring the effects to enhance cell survival in TrkB expressing NB cells. Therefore, we hypothesized that these compounds act as TrkB agonist. We first examined in vitro and in vivo whether the compounds induce TrkB phosphorylation. Western blot analysis in SH-SY5Y/TrkB cells revealed that the treatment of compound 48 and 56 increased phosphorylation of TrkB and its downstream molecules, AKT and ERK. Intraperitoneal injection of compounds 48 and 56 in C57BL/6J mice caused higher phosphorylation of TrkB in hippocampus and cerebral cortex compared with vehicle-injected controls. In SH-SY5Y/TrkB cells treated with the compounds, the phosphorylation of TrkB was blocked by the pretreatment of a Trk inhibitor, K252a. Finally, to investigate the antidepressant effects of the compounds, we carried out the forced swim test（FST）, a model for assessing antidepressant-like behavior. As the result of the FST in C57BL/6J mouse, the both compounds showed reduced immobility compared with vehicle-injected controls. These data suggest that the compounds 48 and 56 could cross the blood brain barrier, activate TrkB and have potential ability of novel antidepressants.		2G2-06 Effects of coffee on vascular endothelial growth factor expression in human neuroblastoma SH-SY5Y cells. Kakio Shota¹，Enoki Soichiro²，Kobata Kenji²，Funakoshi-Tago Megumi¹，Tamura Hiroomi¹ ¹Graduate School of Pharmaceutical Sciences, Keio University，²Graduate School of Pharmaceutical Sciences, Josai University Objectives Coffee is one of the most world-widely consumed beverage on a daily bases. Recent epidemiological studies have reported that amyotrophic lateral sclerosis（ALS）patients are less frequent and prolonged to coffee intake than healthy persons. However, the precise molecular mechanisms of the effects of coffee are yet uncertain. Vascular endothelial growth factor（VEGF）is known to have protective effects on ALS in development of symptoms and prolongation of life. Therefore, we investigated the effects of coffee on VEGF expression in human neuroblastoma SH-SY5Y cells.Methods SH-SY5Y cells were cultured in Ham’s F-12/DMEM（1：1）medium supplemented with 15% FBS. The cells were exposed to coffee or coffee extracts up to at 2.0%（v/v）. After 4 hours, the whole cell lysates were isolated and subjected to immunoblotting for HIF-1α. VEGF gene expression was monitored by qPCR using RNAs isolated from the cells treated with coffee for 8 hours. After 12 hours, the amount of VEGF in the culture medium was measured with an ELISA kit（eBioscience）. Results Coffee induced VEGF expression in dose-dependent manner whereas decaffeinated coffee or caffeine（100 μM）showed no effects. The induction profile of VEGF was corresponding to that of an activation of HIF-1α by coffee. The active constituents of coffee were produced by roasting process of coffee beans and were extractable with n-butanol.Conclusion Coffee induced VEGF expression via the HIF-1α activation in SH-SY5Y cells. This activity may contribute to the preventive effects of coffee on ALS. Further study to identify active components and to elucidate the mechanism of the effects is needed to clarify the molecular basis of neuroprotective effect associated with daily coffee consumption.