Poster

Poster 12 Neurological Diseases 2 ポスター 12 神経疾患2

P12-1 Pathophysiological changes of progranulin in activated microglia after cerebral ischemia 脳梗塞後の活性型ミクログリアにおける progranulin の病態生理学的変化 Horinokita Ichiro（堀之北一朗） School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Tokyo, Japan Progranulin (PGRN) is a cysteine-rich secretory protein which is implicated in neuronal protection, tissue regeneration, and inflammation. In the central nervous system, it has been reported that PGRN protects against neuroinflammation and vascular disorders after cerebral ischemia or hemorrhage. On the other hand, it is known that granulin (GRN), which is cleaved from PGRN by neutrophil elastase, shows pro-inflammatory effects. However, pathophysiological roles of PGRN and GRN after cerebral ischemia have not been fully determined. In this study, we examined time-course of changes in the levels of PGRN and GRN and their cellular sources after cerebral ischemia. A rat microsphere-embolism (ME) model mimicking human multiple infarction was used for in vivo study. In addition, rat neonatal primary cultured microglia, which were isolated from cerebral cortex, were used for in vitro study. Changes in the levels of PGRN and GRN were determined by western blot, qPCR and immunohistochemical analyses. Protein and mRNA levels of PGRN were increased in the ischemic region of cerebral cortex on day 3 after ME. Furthermore, expression of PGRN was increased in activated microglia in ischemic region of ME. Elastase activity in cerebral cortex was increased on day 1 after ME. Protein levels of GRN were increased on days 1 and 3 after ME. These results suggest that increased elastase activity causes cleavage of PGRN, and then GRN may promote inflammatory responses after ischemia. Thus, the changes in the levels of PGRN and GRN might contribute to pathological alterations after cerebral ischemia. It is necessary to clarify the pathophysiological significance of PGRN and GRN in ischemic disorders.		P12-2 Brain water channel AQP4 involvement in normalization of extracellular potassium after acute ischemic stroke 脳虚血超急性期の細胞外カリウムイオン濃度正常化における水チャネル AQP4 の関与 Monai Hiromu（毛内拡）^1,2，矢作和子²，Wang Xiaowen²，阿部陽一郎³，安井正人³，岩井陽一²，平瀬肇² ¹Ochanomizu Univ., Tokyo, Japan ²RIKEN CBS, Wako, Japan ³Keio Univ.,Tokyo, Japan The mechanisms underlying metabolic waste clearance and extracellular ion homeostasis in the brain have not been fully understood. The glymphatic model proposes that extracellular fluid movement in the brain is regulated by aquaporin 4 (AQP4), the water channel expressed in predominantly in astrocytes, and noradrenaline levels (Iliff et al., 2012; Xie et al., 2013). We found that systemic adrenergic receptor blockade facilitates the normalization of extracellular potassium concentration after acute ischemic stroke and mitigates the resultant tissue damage. To test if AQP4 plays a role in this treatment mechanism, we evaluated the expression of AQP4 at various time points after stroke by histology and immunoblot. We employed photothrombosis to induce a local stroke by i.p injection of Rose Bengal and subsequently 15-min green light illumination. For the treated mice, we injected AdR blockers at 30-min before the stroke induction. Mice were perfusion-fixed at 3 hours after photothrombosis induction. AQP4 was stained by the antibody in the coronal section of the cerebral cortex. We found that AQP4 expressions were decreased around photothrombosis induction site, whereas pre-treatment of the AdR blockers preserved AQP4 expressions even after the stroke. This result suggests that the positive effect of AdR blockade on the enhancement of recovery from the stroke could be due to clearance of the extracellular fluid movement regulated by AQP4. We are further investigating how AQP4 is involved in the treatment of the hyperacute phase of the stroke.

P12-3 The role of newly-generated astrocytes and blood vessels in scaffold formation for neuroblasts migrating toward the injured region after ischemic stroke 脳梗塞後の傷害部位への神経前駆細胞の移動のための足場形成に関与する新生アストロサイト・血管の役割 Otake Kyoka（大竹杏佳）¹，岩田英敏^1,2，柴田帝式¹，金子奈穂子¹，澤本和延¹ ¹Dept. Dev. Regen. Biol., Nagoya City Univ. Grad. Sch. Med. Sci., Nagoya, Japan ²Department of Orthopaedic Surgery, Nagoya City University Medical School, Nagoya, Japan Migration of immature new neurons (neuroblasts) from their birthplace in the adult brain, the ventricular-subventricular zone (V-SVZ), toward the olfactory bulb is guided and supported by astrocytes and blood vessels. Ischemic stroke immediately induces gliogenesis and angiogenesis in and around the lesion, followed by migration of neuroblasts from the V-SVZ toward the injured area for neuronal regeneration. However, the role of newly-generated astrocytes and blood vessels as scaffolds for the neuroblasts is largely unknown. Here, using a transgenic mouse line (GFAP-EGFP), we studied the morphology and distribution of newly-generated astrocytes and blood vessels, and their association with the migrating neuroblasts in the post-stroke striatum. The mice just after transient middle cerebral artery occlusion were continuously administrated with BrdU until fixation for 7, 12 or 18 days. By immunohistochemistry, the density and percentage of BrdU-labeled newly-generated cells were quantified separately in the three areas of the striatum: medial intact area, area with reactive astrocytes, and lesion core. In the glial scar and lesion core, the density and the percentage of BrdU+ astrocytes and percentage of BrdU+ blood vessels were significantly higher at day 18 compared with those at day 7. In the 12 and 18 day-post-stroke striatum, most of migrating neuroblasts were observed to be closely associated with BrdU- (pre-existing) blood vessels within the reactive astrocyte-enriched area, suggesting that stroke-induced new vessels support neuroblasts less efficiently. Further characterization of the scaffolds that promote neuroblast migration should contribute to the development of regenerativetherapies for stroke.		P12-4 Effect of Nurr1 ligand amodiaquine on pathology of intracerebral hemorrhage in mice Nurr1 リガンド amodiaquine のマウス脳出血病態に対する作用 Kinoshita Keita（木下慶大）¹，松本倖政²，倉内祐樹²，久恒昭哲^3,4，関貴弘²，香月博志² ¹Dept. of Chemico-Pharmacological Sciences, School of Pharmacy, Univ. of Kumamoto ²Dept. of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Univ. of Kumamoto ³Priority Organization for Innovation and Excellence, Graduate School of Pharmaceutical Sciences, Univ. of Kumamoto ⁴Prog Leading Grad Sch "HIGO Program", Univ. of kumamoto Intracerebral hemorrhage (ICH) is characterized by high mortality and devastating neurological deficits resulting from the formation of hematoma in the brain parenchyma. Several lines of evidence suggest that agonists at nuclear receptors such as RAR and PPARγ produce beneficial effects on the outcome of ICH. Nurr1 (NR4A2) is another member of nuclear receptor family, and in the central nervous system, this receptor is implicated in suppression of pro-inflammatory responses of microglia and astrocytes as well as in the maintenance of survival of midbrain dopaminergic neurons. Here we addressed the possibility that Nurr1 serves as a target for ICH therapy. For this purpose, we used an anti-malarial drug amodiaquine that has been reported to possess agonistic activity on Nurr1. ICH was induced in the striatum of male ICR mice by local injection of type VII collagenase. Amodiaquine (40 mg/kg) was administered intraperitoneally at 3 h after ICH, and thereafter, every 24 h. At 6 h, 1 d, 3 d and 7 d after ICH, motor functions of mice were evaluated by beam-walking test and modified limb placing test. Tissue pathology was examined at 72 h. Expression levels of cytokine/chemokine mRNAs were examined at 6 h after ICH. ICH was accompanied by prominent activation of Iba1-positive microglia/macrophages and GFAP-positive astrocytes in the peri-hematoma region, and also by increased expression of mRNAs encoding IL-1β, CCL2 and CXCL2. These inflammatory responses were markedly attenuated by amodiaquine. Amodiaquine also tended to increase the number of NeuN-positive cells remaining in the hematoma, and improved motor functions of mice at 3 d after ICH induction. These results indicate that Nurr1 modifies several pathogenic events associated with ICH.

P12-5 Changes in expression of BDNF pro-peptide and p75NTR during aging 加齢によるBDNFプロペプチドとp75受容体の発現変化 Matsui Konomi（松井このみ）¹，水井利幸¹，小島正巳^1,2 ¹Molecular and Cellular Pathology Research Team, Biomedical Research Institute, AIST, Osaka, Japan ²Graduate School of Frontier Biosciences, Osaka University, Osaka, Japan Brain-derived neurotrophic factor (BDNF) is the most widely distributed neurotrophin in the central nervous system, and plays many functions such as neural survival, differentiation, and plasticity. Accumulating evidence indicates that reduced BDNF expression in adult brains are associated with the pathogenesis of various brain disorders. The precursor of BDNF (proBDNF) is enzymatically cleaved, by either intracellular or extracellular proteases, to generate mature BDNF (mBDNF) and its pro-peptide (BDNF pro-peptide). Although the expression levels of mBDNF have been shown to decrease in brain tissues with aging, the expression levels of the BDNF pro-peptide throughout the lifespan are poorly known. In this study, we analyzed biochemically the expression levels of the BDNF pro-peptide in aged-mouse brain tissues and compared with the changes of mBDNF expression and the BDNF pro-peptide in older adult mice.The expression levels of mBDNF in both hippocampus and frontal cortex gradually decreased during aging. The significant age-related decline observed in 12-month-old mice (40% reduction, n = 3). Interestingly, the expression levels of BDNF pro-peptide in frontal cortex gradually increased during aging. The significant age-related upregulation observed in 12-month-old mice (30% increase, n = 3). In contrast, the expression levels of BDNF pro-peptide in hippocampus gradually decreased during aging similarly to the mBDNF expression. Finally, we found that the expression levels of pan-neurotrophin receptor p75NTR in frontal cortex increased during aging. These results indicate that the BDNF pro-peptide and p75NTR were up-regulated in frontal cortex during aging.		P12-6 Quantitative analysis of myeloperoxidase (MPO) and brain-derived neurotrophic factor (BDNF) in plasma and saliva in aged 老化に伴う神経変性のリスク評価法開発の試み：ミエロペルオキシダーゼタンパク測定と活性比較を中心として Shamoto-Nagai Masayo（永井雅代）¹，西口寛一郎²，小島規永²，武部純²，直井信¹，丸山和佳子¹ ¹Dept. of Health and Nutrition, Faculty of Psycological & Physical Science , Aichi Gakuin Univ. ²Dept. of Removable Prosthontics, School of Dentistry, Aichi Gakuin University Aging is the most common and major risk factor of neurodegenerative disease but its mechanism has not been well elucidated. However, chronic inflammation and increased oxidative stress are suggested to be involved. Periodontal disease is now gathering attention as a cause of systemic inflammation. Increased plasma myeloperoxidase (MPO) level is reported in neurodegenerative disorders such as Alzheimer disease and Parkinson disease. MPO protein is released into extracellular fluid during inflammation from neutrophils, etc. to induce oxidative damage. The protein levels MPO in plasma and saliva samples were estimated. In addition, plasma MPO activity was quantified also. The subjects of the study were twenty-six patients (F=22, M=4) aged 88.2 ± 9.5 (52-101) y.o. The plasma samples were obtained from twenty-six patients and the saliva samples were from fifteen patients. MPO protein content in the plasma and the saliva using ELSA. Plasma MPO activity in the plasma was quantified by neutrophil MPO activity kit.Plasma MPO content level was variable from 2.14 ng/ml to 92.9 ng/ml and saliva MPO content was from 37.5 ng/ml to 2980 ng/ml. On the other hand, plasma MPO activity was not so variable from 3.71 unit to 33.1 unit (abs/min/μl). MPO content in the saliva was largely dependent on the clinical severity of periodontal disease. However, saliva MPO content did not affect plasma MPO content significantly but further studies with larger number of patients are needed. Plasma MPO content did not correlate with plasma MPO activity. It might indicate intrinsic MPO inhibitor regulate plasma MPO activity. Further study is needed to find out biological marker of ageing and age-related neurodegeneration.

P12-7 Traumatic brain injury in aged Drosophila induces hyperactivation of innate immunity ショウジョウバエ老化個体における外傷性脳損傷は過剰な自然免疫応答を惹起する Tanaka Tomoya（田中智也）¹，茨木公英¹，佐貫理佳子^1,2 ¹Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan ²Department of Drosophila Genomics and Genetic Resources, Center for Advanced Insect Research Promotion, Kyoto Institute of Technology, Kyoto, Japan Penetrating traumatic brain injury (pTBI) is a direct injury in the brain parenchyma occurs by an object penetrating to the brain as like a gunshot. It has been reported that one of the significant prognostic factors for mortality in pTBI patients after a gunshot wound is age older than 35 years. In these days, novel animal models to understand pTBI have been developed with rodents, however, it is still difficult to analyze the relationship between an increased mortality and aging in pTBI because of a difficulty in getting aged animals.The fruit fly Drosophila melanogaster is one of the most useful model organisms with a short life cycle of approximately two weeks and has a far shorter lifespan than that of mammals. Therefore, Drosophila is attractive for studying aging. In this study, to examine the molecular basis behind the increased mortality by aging in pTBI, we developed a novel pTBI model using Drosophila.First, we confirmed that aged flies showed higher mortality than young flies by pTBI, which is similar to human studies. We then found hyperactivation of genes involved in innate immunity in the pTBI-treated aged heads. We further analyzed expression of genes on immune pathways and found that in the aged pTBI flies, compared to the young pTBI flies, the IMD pathway was highly activated, but the other pathways were downregulated. Our results suggest that this unbalanced hyperactivation among the immune pathways caused by aging could be one of the reasons for high mortality in aged individuals with pTBI.		P12-8 Pathophysiological analysis of inherited leukodystrophy with defective myeline lipid metabolism and its therapeutic application to the demyelinating diseases Myelin lipid 分解経路の破綻がもたらす脳白質障害の病態解析とその治療応用 Enokido Yasushi（榎戸靖）¹，鬼頭もも子²，郷慎司³，細川昌則¹，浅井清文²，竹林浩秀⁴，松田純子³，稲村直子¹ ¹Dept. of Pathol., Inst. of Dev. Res., Aichi Human Service Center ²Inst. Mol. Med. Grad. Sch. of Med., Nagoya City Univ. ³Dept. of Pathophys. & Metab, Kawasaki Med. Sch. ⁴Grad. Sch. of Med. and Dent. Sci., Niigata Univ. Myelin is a sheath-like structure wrapping around the nerve axon. It is formed by an electrically insulating membranes that allows to propagate saltatory conduction of nerve impulse. The primary lipid of myelin is a glycolipid called galactosylceramide (or galactocerebroside), which is catalyzed by a lysosomal enzyme galactosylceramidase (GALC). Mutations in GALC gene causes Krabbe disease (KD), one of the lysosomal storage diseases with inherited leukodystrophy. The majority of KD patients are characterized by infantile-onset cerebral demyelination with apoptotic oligodendrocyte (OL) death. Currently, there are no effective treatments for KD. In the present study, we investigated cellular mechanisms of pathogenic changes in developing KD OLs by using twitcher mice, an authentic mouse model of KD. In twitcher mouse brain, myelination was impaired, and the myelin gene and protein expression were significantly decreased when the period of active myelination prior to pathogenic demyelination. To investigate the differentiation and maturation of KD OLs in detail, we cultured primary OL precursor cells (OPCs) isolated from twitcher mouse brains. Interestingly, twitcher OPCs proliferated normally, but their differentiation and survival were intrinsically defective. These defects were associated with abnormally accumulated endogenous psychosine (galactosylsphingosine), and the reduced activation of some signaling pathways involved in OL differentiation and maturation.