Poster Sessions

Poster Sessions ISN-APSN-JSN Advanced School

2P-64 The roles of sphingosine 1-phosphate signaling in blood brain barrier integrity Ping Xiang Department of Pharmacology, Yong Loo Lin School of Medicine, National University of Singapore Disruption of blood brain barrier (BBB) integrity has been found in a number of neuropathological diseases, including vascular dementia, stroke and multiple sclerosis. Studies have shown that modulation of sphingosine-1-phosphate (S1P) signaling pathway is emerging as potent target of vascular integrity via its receptors. S1P receptor subtype 1 (S1PR1) has been shown to promote endothelial tight junction integrity, while S1PR2 seems to disrupt BBB properties. Besides endothelial cells, S1P receptors are also functionally expressed in astrocytes, which are one of the main compositions of BBB. However, how modulation of S1P signaling in astrocytes affect the BBB integrity is still not clear yet. In this study, primary astrocytes were purified from neonatal mouse pups P0-3. Pharmacological manipulation of S1P signaling pathway with selective agonists and antagonists of different subtypes of S1P receptors (S1PRs) were applied to primary astrocytes. The effects of astrocyte-conditioned medium in endothelial barrier integrity were further investigated by western blot and immunocytochemistry with tight junction proteins, including Claudin-5 and ZO-1. Our results have shown that astrocytes mainly expressed S1PR1, S1PR2 and S1PR3 receptors along with other subtypes at a very low level. S1P triggered an upregulation in mRNA expression of cytokines as well neuroprotective factors such as BDNF and GDNF in astrocytes. However, S1P receptors play a differential role in the cytokine expression files of astrocytes. Activation of S1PR2 promotes the expression of pro-inflammatory cytokines, while activation of S1PR1 and S1PR3 shows an anti-inflammatory role. It suggests that S1P receptors may play a differential role in BBB integrity through modulation of the secreted factors from astrocytes.		2P-65 Dihydromyricetin protects neurons in an MPTP-induced model of Parkinson’s disease and cerebral ischemia/reperfusion injury Zhaoxiang Ren College of Pharmaceutical Sciences, Soochow University Dihydromyricetin (DHM), a natural flavonoid compound extracted from the fruit Ampelopsis grossedentata, exerts various pharmacological effects. DHM has been shown to be a potent anti-oxidative agent and to protect mitochondrial functions. In addition, recent studies have reported that DHM has anti-tumor and anti-inflammatory effects. Interestingly, DHM can also protect PC12 cells from oxidative stress and elicit beneficial effects in both Alzheimer's disease and alcohol intoxication. Therefore, we explored the neuroprotective effects of DHM following cerebral ischemia as well as in an MPTP-induced model of Parkinson’s disease.Male mice were intraperitoneally injected with 1-methyl4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) for 7 d to induce PD and middle cerebral artery occlusion (MCAO) was performed. Behavioral tests were administered after DHM treatment and brain tissue was collected for immunofluorescence staining and Western blotting. In addition, neuron cell lines were treated with DHM, and evaluated using cell viability assays, reactive oxygen species (ROS) measurements and apoptosis analysis. Pretreatment of DHM significantly attenuated MPTP-induced mouse behavioral impairments and dopaminergic neuron loss by suppressing glycogen synthase kinase-3 beta activity. Moreover, DHM reduced the activation of microglia, protected HT22 neurons against OGD-induced injury and promoted functional recovery following middle cerebral artery occlusion (MCAO) by suppressing microglial neuroinflammation and activating neuronal ERK-CREB-Bcl-2 signaling pathway.

2P-66 IFITM3 regulates polyI:C-induced neurite impairment through RabGDI Norimichi Itoh Nagoya University Graduate School of Medicine/Hospital Pharmacy and Neuropsychopharmacology Perinatal viral infection is one of the environmental risk factors for the psychiatric disorders, such as schizophrenia, autism and bipolar disorder. We have reported that treatment of polyriboioinic-polyribocytidylic acid (poly I:C), a synthetic double strand RNA which induces natural immune reaction, in neonatal mice lead to schizophrenia-like behavioral abnormality in adulthood. We have found interferon-induced transmembrane protein (Ifitm3) in astrocytes as the key molecule in poly I:C-induced behavioral alteration. However, the molecular mechanism underlying roles of Ifitm3 in CNS remains unknown. To understand how Ifitm3 causes developmental abnormalities in CNS, we explored novel Ifitm3-iteracting proteins by proteomic approach. As a result, we identified RabGDI as a novel Ifitm3-interactig protein. Interaction of Ifitm3 with RabGDI was validated by immunoprecipitation and GST pull down assay in HEK293FT cells. Localization of Ifitm3 was partially merged with RabGDI. Expression of Ifitm3 increased size of EEA1-positive endosome. GST pull down assay revealed that Ifitm3 increased level of active Rab5. Treatment of condition medium from dominant negative Rab5 expressing astrocyte partially rescued the polyI:C-induced inhibition of neurite elongation. These results suggest that Ifitm3 mediates polyI:C-induced neurite impairment by modulating Rab5 activity.		2P-67 Phosphorylation CaMKII and ERK may mediate nicotine dependence in mice Gofarana Wilar^1,2，Yasuharu Shinoda¹，Kohji Fukunaga¹ ¹Dept Pharmacol. Grad Sch Pharm Sci, Tohoku Univ，²Faculty of Pharmacy, Padjadjaran University, Bandung, Indonesia Nicotine is an active compound and has a rewarding effect in the CNS, which may leads to dependence. Although nicotine dependence is elucidated by brain mechanisms, synaptic molecular substrates underlying the dependence remains unclear. We hypothesized that reward signaling is mediated by dopamine and glutamate receptors, in where CaMKII and ERK may mediate the synaptic signaling of dependence. To investigate the roles of both CaMKII and ERK on nicotine dependence, we first measured CaMKII and ERK phospohyrlation after establishment of nicotine dependence assessed by conditioned placed preference (CPP). Mice were first habituated to the CPP apparatus for five days, followed by a pre-conditioning test to determine the nicotine-paired compartment. Mouse entered conditioning training for one month in which 0.5 mg/kg nicotine was administered intraperitoneally followed by confinement in the designated compartment of CPP apparatus for 30 minutes. Four hours later, the same procedure was repeated, only this time saline was given instead of nicotine and the mouse was confined in the opposite of nicotine compartment. One day after conditioning, preference scores were measured to evaluate the nicotine dependence. Mice were sacrificed and their striatum were dissected out for immunoblotting analysis of CaMKII and ERK phosphorylation. CaMKII and ERK phosphorylation significantly increased along with development of nicotine dependence. We should next apply pharmacological strategies to manipulate CaMKII and ERK signaling. In particular, disruption of reconsolidation by disrupting CaMKII and ERK signaling may propose an attractive therapeutic approach to inhibit nicotine dependence.

2P-68 Phosphorylation of tau from Marmoset used as a Non-human Primate model of tauopathy. Govinda Sharma¹，Taeko Kimura¹，Shiozawa Seiji ²，Reona Kobayashi²，Taro Saito¹，Kanae Ando¹，Hideyuki Okano²，Shin-ichi Hisanaga¹ ¹Dept Biological sciences, Tokyo Metropolitan University，²Department of Physiology, Keio University School of Medicine Tau hyperphosphorylation, a hallmark of Alzheimer’s disease, is found in other neurodegenerative diseases collectively called tauopathy. However, it is not known why and how tau is hyperphosphorylated in AD or tauopathy brains. There are unavoidable limitations in using patient’s brains, particularly dephosphorylation during post mortem interval is problematic. Although tau pathology has been studied using transgenic rodent models, there are no rodent models reflecting AD pathology exactly. Marmosets, which are phylogenetically closer to humans, have recently been used as a model for neurodegenerative diseases. Marmosets are preferred over other primates due to their shorter lifespan, small size and capability of gene engineering. While it has been reported that tau hyperphosphorylation is detected histochemically in aged marmosets, there is no biochemical report yet. In this study, we cloned tau cDNA from the cDNA library of adult marmoset brain. They expressed, at least, two isoforms of tau: 0N4R and 2N4R, and the 2N4R isoform of marmoset tau showed 94.34 % homology to the respective human tau isoform. Tau was detected as two major bands in marmoset brain with similar electrophoretic mobility to human 0N4R and 2N4R tau. These two isoforms were equally expressed in hippocampus, striatum and cortex, but in olfactory bulb 0N4R tau was predominant, while in cerebellum 2N4R was predominant. These electrophoretic profiles of tau showed resemblance to those of mouse brains more than that of human. Levels of phosphorylation were higher in marmoset than mouse or human brain. These results provide valuable biochemical information on tau pathology, because unlike in mouse, tau is physiologically hyperphosphorylated in adult marmoset brain.		2P-69 New analogs of oxytocin. In vitro activity, specificity and effects on parental behaviour. Stanislav Cherepanov¹，Anna Shabalova¹，Wataru Ichinose²，Satoshi Shuto²，Shigeru Yokoyama¹，Haruhiro Higashida¹ ¹Research Center for Child Mental Development, Kanazawa University，²Faculty of Pharmaceutical Sciences, Hokkaido University Autism spectrum disorder (ASD) is a neurodevelopmental disorder characterized by impairments in social communication and repetitive behaviors. There are few effective treatments for the symptoms of ASD. A great deal of research has focused on the promising candidate neuroendocrine hormone, oxytocin (OT), for the treatment of social deficits. However, OT has a short time of half-life in blood and high affinity not only to OT-receptor (OTR) but also to vasopressin receptor -V1A and V1B-subtypes. Analogs of OT with long-lasting effect and high selectivity should have a benefit as a therapeutic drug. According to this, we newly designed and synthesized two oxytocin analogs: X1 and X2, other than our previous lipidated OT called LOT-1, 2 and 3 (Cherepanov et al.; 2017). As in vitro experiments, we investigated release of intracellular calcium in stable transfected cell lines, expressed OTR- or V1AR. X1 increased calcium concentration with similar affinity as OT to OTR and X2 slightly smaller one. Comparing with OT both had significantly lower affinity to V1A-receptors. We investigated OT and two analogs on paternal behavior tests with CD38-/- mice. In the parental behavior test, CD38-/- male mice demonstrate low pups retrieving scores. In the case of OT, X1 or X2, at 30 minutes after intraperitoneal injection, CD38-/- male mice increased retrieving scores. After 3-24 hours, for OT treated mice scores were as low as that in non-treated CD38-/- mice, X1 and X2 demonstrated higher effects on parental scores.This result indicates that two novel OT analogs have high affinity to OT-receptors, with a high selectivity and possess immediate and long-lasting effects, suggesting that they are useful for recovery of social behavioral impairment.

2P-70 The developmental neurotoxicity of arsenic: cholinergic, dopaminergic dysfunctions with cognitive, behavioral consequences of perinatal exposure Lalit Chandravanshi Institute of Science, Department of Zoology, Banaras Hindu University The majority of recent occupational studies focused on environmental toxicants, which were associated with neurobehavioral impairments in the progeny. Additional evidence on environmental exposures demonstrates the vulnerability of the developing brain to arsenic (As). There is much less information on effects of As on developing brain. We hypothesized that the As induced neurotoxicity may reversible or not after withdrawal of following As exposure (2 mg/kg and 4 mg/kg body weight, p.o.) in perinatal developing period of brain of rats. Rats were exposed to As from GD06 to PD21 and one set of rat left for withdrawal of exposure from PD22 to PD45. Effect on cholinergic and dopaminergic signaling in different brain regions of rats along with behavioral modifications and also developments of physical milestones or reflexes were studied.Perinatal exposure to As resulted to impair surface righting, cliff avoidance response and forward locomotion with head up position and the changes were significant in rat pups exposed to As. In this study we were observed the decrease forelimb grip strength, alter learning and memory in As exposed rats at both doses as compared to controls. As exposure caused a significant decrease in the expression of CHRM2 receptor gene were observed in frontal cortex and hippocampus of rats on PD22 as compared to controls. Acetylcholinesterase activity, density of Nissl granules in the dentate gyrus area of hippocampus and expression of ChAT and PKCβ-1 were also altered in As exposed rats.Motor activity was decreased in As exposed groups remarkably compared to control and associated with decrease in the binding of DA-D2 receptors or the expression of tyrosine hydroxilase (TH), DARPP32 and DAT in the corpus striatum. Further, significant decrease in the mRNA level of DAR-D2, or dopamine and its metabolites (DOPAC and HVA) content were also observed suggesting impaired dopaminergic signaling. While a trend of recovery was found in cholinergic and dopaminergic system in brain but changes remained persistent after the withdrawal of As exposure on PD45 in comparison to controls.Findings of these experiments suggest that perinatal As exposure appears to be critical and vulnerable as development of cholinergic and dopaminergic system continues during this period.		2P-71 Stearic acid based, oral lipidic nanoparticles of dimethyl fumarate and quercetin for the management of relapsing multiple sclerosis: enhanced pharmacokinetics and pharmacodynamic profile Pramod Kumar Department of Pharmacy, School of Chemical Sciences and Pharmacy, Central University of Rajasthan Almost 2.5 million people have been affected by multiple sclerosis worldwide including India in the last decade. Dimethyl Fumarate is frequently available in the market in the different doses for the treatment of relapsing multiple sclerosis. It is associated with issues like multiple dosing, gastric intolerance and poor brain permeability. It is envisioned to develop a once-a-day formulation along with quercetin with enhanced brain permeability and improved gastric compatibility. Pre-screening of lipids (06 formulations), screening of the formulation attributes (08 formulations) and optimization (20 formulations) of the formulation were performed and validated with particle size, drug release and entrapment efficiency using design expert software trial version 9.0.3.1. Optimized solid lipid nanoparticles were altered and formula was redesigned for quercetin tagging and characterized for entrapment, drug loading and in-vitro release studies. Caco-2 and SH-SY5Y cellular uptake studies were performed. In-vivo Pharmacokinetic and biodistribution studies in Wistar rats were also done. Cuprizone induced demyelination were employed to perform behavioural and histopathological studies. Luxol fast blue dye was used to stain the demyelination and remyelination part in brain. Particle size, zeta potential and PDI for DMF-quercetin loaded solid lipid nanoparticles was found to be 122.76± 4.91, -3.66 and 0.377, respectively. Entrapment efficiency and drug loading was observed 86.32 ± 2.59 and 22.07 ± 0.67, respectively. Sustain release with Higuchian profile was observed to that of pure drug. Enhanced oral bioavailability, caco-2 and SH-SY5Y cellular permeability, and GIT compatibility were found to be superior to that of conventional formulations. Enhanced brain permeability was observed in in-vivo studies. Behavioural functions like grip strength and motor co-ordination of the Laca mice for optimized formulation are superior than pure drug and disease induced group. Enhanced remyelination and bioavailability, brain and intestinal permeability and GIT compatibility is proved that solid lipid nanoparticles along with quercetin are the better option for relapsing multiple sclerosis. SLNs with quercetin may be a promising alternative carrier for management of neurological disorders like relapsing relapsing multiple sclerosis employing DMF.

2P-72 Potential effects and molecular mechanisms of melatonin on the dopaminergic neural differentiation of human amniotic fluid mesenchymal stem cells Ruchee Phonchai Research Center for Neuroscience, Institute of Molecular Biosciences, Mahidol University Melatonin is secretory hormone mainly from pineal gland in the brain which has physiological functions and neuroprotective effects. Previous studies reported that melatonin exert its effect on mesenchymal stem cells (MSCs) differentiate into osteocgenic- and adipogenic-lineage. However, the effect and mechanism of melatonin to induce neurogenic differentiation in MSCs are unclear. Thus, we aimed to investigate the potential effects and molecular mechanisms of melatonin on dopaminergic neuron differentiation of amniotic fluid (AF)-MSCs. Stem cells were isolated from amniotic fluid and cultured for expansion and MSCs characterization. AF-MSCs were sequential induced in neurobasal medium containing standard cocktails (bFGF, SHH, FGF8, BDNF), 1000 nM melatonin, or a combination of standard cocktails and melatonin for 12 days. Flow cytometry analysis showed no different in the positive cells of NCAM and phosphorylation of TH (phospho-TH) in vehicle (V)-, standard cocktails (ST)-, melatonin (M)- and ST plus M inducing AF-MSCs. CD133 positive cells were significantly decreased in V, ST, M and ST plus M inducing AF-MSCs. The western blot analysis showed significantly increased in the levels of TUJ1, TH, MAP2, NURR1 and dopamine transporter (DAT) in ST, M and ST plus M inducing AF-MSCs compared with V group. In addition, the levels of DAT in ST plus M inducing AF-MSCs was significantly increased compared with M group. The MT1 melatonin receptor was tended to decrease in ST, M and ST plus M inducing AF-MSCs compared with V group. Melatonin significantly increased MAP2 and pERK levels in AF-MSCs when compared with vehicle group and this effect can be inhibited by pretreatment with luzindole, a melatonin receptor antagonist. However, melatonin-induced increase in pCaMKII was not blocked by luzindole pretreatment. In conclusion, In conclusion, In conclusion, In conclusion, In conclusion, In conclusion, melatonin promotes the differentiation of AF-MSCs into neurons via the induction in ERK pathway through MT1 receptor activation. CaMKII pathway may also involve in the differentiation process but not mediate through MT1 receptor-dependent mechanism.		2P-73 nArgBP2 regulates excitatory synapse formation by controlling dendritic spine morphology Sang-Eun Lee College of Medicine / Biomedical Sciences, Seoul National University Neural Abelson-related gene-binding protein 2 (nArgBP2) was originally identified as a protein that directly interacts with synapse associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3), a postsynaptic scaffolding protein critical for the assembly of glutamatergic synapses. Although genetic deletion of nArgBP2 in mice leads to manic/bipolar-like behaviors resembling many aspects of symptoms in patients with bipolar disorder, the actual function of nArgBP2 at the synapse is completely unknown.Here, we found that the knockdown (KD) of nArgBP2 by specific small hairpin RNAs (shRNAs) resulted in a dramatic change in dendritic spine morphology. Reintroducing shRNA-resistant nArgBP2 reversed these defects. In particular, nArgBP2 KD impaired spine synapse formation such that excitatory synapses terminated mostly at dendritic shafts instead of spine heads in spiny neurons, although inhibitory synapse formation was not affected. nArgBP2 KD further caused a marked increase of actin cytoskeleton dynamics in spines, which was associated with increased Wiskott–Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE1)/p21-activated kinase (PAK) phosphorylation and reduced activity of cofilin. These effects of nArgBP2 KD in spines were rescued by inhibiting PAK or activating cofilin combined with sequestration of WAVE. Together, our results suggest that nArgBP2 functions to regulate spine morphogenesis and subsequent spine-synapse formation at glutamatergic synapses. They also raise the possibility that the aberrant regulation of synaptic actin filaments caused by reduced nArgBP2 expression may contribute to the manifestation of the synaptic dysfunction observed in manic/bipolar disorder.

2P-74 Stat3 Promotes Mitochondrial Metabolism during Neurogenesis Yixun Su Neurobiology and Aging Program, Life Science Institute, National University of Singapore, Centre for Life Sciences STAT3 plays a key role in neural progenitors during embryonic development. It was reported that STAT3 regulates expression of astrocyte markers such as GFAP and S100beta, and that STAT3 maintain the stemness of radial glia at the mid-neurogenesis stage. However, the absence of transcriptome study limits our understanding of the function of STAT3 in neural progenitors. Here we identified potential Stat3 target genes by using RNA-seq to compare expression profiles of WT and Stat3 conditional knockout neural progenitors. We found that STAT3 directly promotes expression of genes that encode subunits of mitochondrial electron transport chain complex, and it promotes mitochondrial respiration during neural progenitor development. Mitochondrial membrane potential and ROS production are also regulated by STAT3. Last but not least, AMPK pathway and mTOR pathway, which are closely regulated by energy metabolism, are also altered in Stat3 conditional knockout neural progenitors, yet the mechanism remains to be elucidated.		2P-75 Prefrontal cortex mediated executive impairment in ADHD may be contributed by functional DRD4 and DAT variants Subhamita Maitra Biomedical Research & Diagnostic Division, Manovikas Kendra Probands with Attention deficit hyperactivity disorder (ADHD) draws attention chiefly due to inattention and hyperactivity/impulsivity. Inter-related domains of executive function (EF), including focussing, optimum effort, emotion control, working memory and prompt action are affected in the probands, indicating EF dysfunction as a major trait. Proper activation of the prefrontal cortex (PFC) is a vital modulator of EF and brain imaging studies exhibited delayed cortical maturation in the ADHD probands. The dopamine receptor 4 (DRD4) is consistently expressed in the PFC while the dopamine transporter (DAT) is expressed at higher level during the post-natal as well as the developing period. Based on these facts, we analyzed association of 15 functional variants of DRD4 and DAT genes with IQ, inattention and EF deficit of ADHD probands. Method: Probands (N=218) were recruited following DSM-IV-TR. Inattention (IA) was measured through Conner’s Parent Rating Scale-revised (CPRS-R) as well as DSM IV-TR. IQ was assessed through Wechsler Intelligence Scale for Children. Short attention span (SAS) and erratic organisational capability (EOC) was measured through DSM-IV-TR and CPRS-R. Barkley Executive Functioning Scale as well as computerised games were used for measuring EF. Peripheral blood collected after obtaining informed written consent was used for genomic DNA isolation and amplification of target sites, followed by genotyping through DNA sequencing or gel electrophoresis. Result: Linear correlation was noticed for SAS, EOC, EF & EF summary of EF (R>0.37, P<0.001). EOC showed a strong positive correlation with age (R=0.20, P=0.004). rs28382221 “G/GG” was found to impart negative influence on SAS, EF summary and EF count (P<0.05). rs2254408 “GT” influenced EF summary score and EF count. DRD4 Exon3 VNTR 7R allele was associated with increase in SAS and EF (P<0.05). rs916455 “CT” showed a trend for negative influence on each parameter. Conclusion: This pilot study investigating the role of functional gene variants in the EF deficit of Indian ADHD probands indicate that differential expression of DRD4 and DAT, originating from polymorphic variations, may lead to altered neurotransmission in the PFC culminating in executive impairment, warranting further in depth investigation.

2P-76 Ginkgo biloba extract provide protection against sleep deprivation induced cognitive deficit. Vishal Jain Defence Institute of Physiology and Allied Sciences Sleep deprivation (SD) is a condition in which the body is deprived of adequate sleep required. Sleep affects the body in a number of ways such as lowering the ability to make decisions, less attentiveness, affecting the working memory and long term memory. The effects include disturbance in neural communications (Synaptic plasticity), decrease the cognition and reduces memory performance. The aim of present study is to evaluate the effect of Ginkgo biloba (GB) on sleep deprivation induced cognitive deficit and synaptic plasticity. Adult male Sprague–Dawley rats (220–240 g) were used as model and sleep deprivation was induced in an in-house made novel chamber. Rats were divided into four different groups as follows: Control, Control + GB, SD (72 hr) and SD (72 hr.) + GB. GB extract was administered orally at a dose of dosage of 100mg / kg body weight (dissolved in saline) for one month before exposure to SD. A light/dark cycle of 12 hr. was maintained at controlled temperature (26 ± 2 ºC) and humidity (50 ± 5%). Behavioural studied was done using OFT, EPM, and MWM apparatus during the light phase between 10:00 to 11:00 am before and after the sleep deprivation. Following exposure and behaviour studies hippocampus was collected and processed for further experiments. Hippocampal coronal sections were used to study the neurodegeneration, level of synaptic proteins and neurogenesis through immunohistochemistry (IHC). Results of the present study showed that SD induces cognitive decline as evident from decreased performance in MWM and decreased exploratory activity as shown in EPM and OFT. Also the level of synaptic proteins decreased significantly on SD exposure with increased neurodegeneration. On the other hand, GB showed promising effect against SD induced cognitive decline, neurodegeneration and loss in synaptic functions. In addition to this it also showed neurogenic effect and increased neuronal activity following GB treatment before SD exposure.