Poster

Poster 4 Neurological Diseases 1 ポスター 4 神経疾患1

P4-1 The effect of ar-turmerone on neuronal survival and axonal growth ar-ターメロンの神経生存保護作用及び軸索伸長能の解析 Saga Yuya（佐賀友弥）¹，畠中雄大¹，吉岡百合³，松村普一³，財満信宏⁴，小西慶幸^1,2 ¹Facl. Eng., Univ. Fukui ²Life Science Innovation Center, University of Fukui ³INABATA KORYO CO.,LTD. Natl. products ⁴Department of Agriculture, Kindai University Previous studies have demonstrated the neuroprotective function of curcumin, a component of curcuma longa (turmeric). On the other hand, the function of ar-turmerone, a main component of sesquiterpene contained in turmeric, in neurons has not been fully studied. In study, we analyzed the function of ar-turmerone in culture of cerebellar granule neurons (CGNs). Firstly, we examined the neuroprotective function of ar-turmerone upon the suppression of neuronal activity by depriving KCl. As a result, 36.0% of neurons were protected from activity deprivation by treating with 50μg/ml of ar-turmerone. Indeed, ar-turmerone reduced the number of activated Caspase-3 positive cells by 54.3% under low KCl condition. Next, we examined the neuroprotective function of ar-turmerone in the presence of glutamate. We found that ar-turmerone administration increased survival rate by 28.6% in the presence of 100μM glutamate. Furthermore, we observed that ar-turmerone increased the length of CGN axons.Together, our study revealed a novel function of ar-turmerone in protecting neurons from apoptosis and enhancing the growth of axons.		P4-2 A molecular signal that controls tissue damage and repair in the brain white matter 白質の損傷と修復を制御するミクログリアの分子シグナル Nagai Hiromi（永井裕美）¹，橋本美穂¹，堀越絢乃¹，Ruwaida Elhanbaly²，石川達也²，的崎尚³，深澤有吾²，大西浩史¹ ¹Dept. Lab. Sci., Gunma Univ. Grad. Sch. Health Sci., Gunma, Japan ²Div. Brain Struct. Func., Fac. Med. Sci., Univ. of Fukui, Fukui, Japan ³Dept. Biochem. Mol. Biol., Kobe Univ. Grad. Sch. Med., Kobe, Japan SIRPα (Signal regulatory protein α) is a member of immunoglobulin superfamily membrane proteins. SIRPα on phagocytes, such as macrophages, specifically interacts with CD47, another membrane protein on phagocytic targets, and this cell-cell interaction signal negatively regulates phagocytosis. In the central nervous system, SIRPα and CD47 are dominantly expressed in matured neurons. SIRPα is also expressed in microglia, the tissue macrophages of the brain, which play a crucial role in phagocytic clearance of damaged brain tissue. Compared to peripheral macrophages, physiological role of CD47-SIRPα signal in regulation of the brain microglia has not been fully understood. We analyzed the effect of genetic ablation of SIRPα on activation of microglia in mouse brain. Lack of SIRPα resulted in an increase in the number of a subpopulation of activated microglia in the brain white matter region. We found that SIRPα deficiency ameliorates demyelination damage in mouse model system. Furthermore, knockdown of SIRPα in microglia model cells resulted in increased phagocytosis of myelin fragment in vitro. Our data suggest that CD47-SIRPα signal is important for the regulation of microglial phagocytosis of myelin fragment and for the control of microglial activation in the brain.

P4-3 IVIg treatment is effective for the Lysolecithin-induced peripheral nerve demyelination of mice マウス末梢神経リゾレシチン誘導脱髄へのIVIg製剤の効果 Hayashi Akiko（林明子），鈴木尚穂，瀬戸口潔，金子真之亮，田中大夢，柳岡大悟，馬場広子 Department of Molecular Neurobiology, Tokyo University of Pharmacy and Life Sciences, Japan Intravenous IgG Kenketu glovenin(R)-I (IVIg) treatment is known to be effective on human immune-mediated neurophathies. IVIg treatment also ameliorates clinical score and conduction velocity in rat experimental autoimmune neurophathy (EAN). However the mechanisms of IVIg teratment in the peripheral nerve demyelination are still uncertain. To elucidate this mechanism, the effect of IVIg on lysolecithin-induced chemical demyelination was examined.ICR mice were intraneurally injected with 1% Lysorecithin into the sciatic nerves, and either IVIg or control saline was injected intravenously 24 hours later. Sciatic nerves were resected, and evaluations of demyelinating areas and infiltrating macrophages were performed. On the day 7 after lysolecithin injection, demyelinating areas of IVIg injected group were smaller than those of saline injected group. Numbers of CD68+Arginase1+ M2 macrophages were increased in IVIg injected group, while CD68+Arginase1- macrophages were increased in saline treated group. The dose of human IgG (glovenin) in the mice sera was detected in enough level even 14 days after the injection. Depositions of human IgG were observed on myelin debris in the demyelinating lesions. By western blotting using IVIg, only a 36-kDa single band of large myelin protein zero (L-MPZ) was detected in rat sciatic nerve homogenates, suggesting that antibodies against L-MPZ in IVIg directly bound to the debris. These results indicate that IVIg treatment has protective effect on chemical demyelinating condition, and inducible effect of M2 macrophages in acute phase. Direct binding of IVIg to myelin debris may be involved in this modulation process.		P4-4 Involvement of gene expression changes via DNA and histone hypomethylation in folate deficiency-induced abnormal neuronal maturation 葉酸欠乏性の神経成熟異常におけるDNA/ヒストンメチル化の関与 Nishida Shoji（西田将治）¹，竹村凌¹，浅利颯太¹，橘新¹，壷井美里²，中村吉孝²，荒木良太¹，矢部武士¹ ¹Laboratory of Functional Biomolecules and Chemical Pharmacology, Faculty of Pharmaceutical Sciences, Setsunan University ²Food Science & Technology Research Laboratories, R & D Division, Meiji Co., Ltd. Folate serves to transfer one-carbon units in various biological reactions such as DNA and histone methylation; therefore folate is an essential nutrition in the development and maintenance of biological function. Our previous animal studies have shown that folate-deficient mice show depression-like behavior and abnormal neuronal maturation in the dentate gyrus. These studies suggest that the abnormal neuronal maturation is a key phenomenon in folate deficiency-induced depressive symptoms. In this study, to investigate the mechanisms of folate deficiency-induced abnormal neuronal maturation, we analyzed mRNA expression and epigenetic modification for neuronal maturation-related genes in neural stem cells derived from fetal mouse brain. The increase in the number of neurons (Tuj1 positive cells) and decrease in the number of mature neurons (MAP2 positive cells) from differentiated cultured neural stem cells were observed under folate-deficient condition for 7 days. Furthermore, mRNA expression for mature neuron-related genes such as Neurod1 and Creb1 was decreased in cultured neural stem cells under the folate-deficient condition. By contrast, mRNA expression for immature neuron-related genes such as Neurog1 and Eomes was increased in these cells. Dot blot and Western blot analyses revealed that DNA methylation and histone H3K27 tri-methylation were decreased in cultured neural stem cells under folate-deficient condition, respectively. In contrast to histone H3K27, histone H3K4, H3K9 and H3K36 tri-methylation levels remained unchanged. These results suggest that folate deficiency-induced abnormal neuronal maturation may be caused by alteration in mRNA expression for neuronal maturation-related genes via epigenetic modification such as DNA and histone hypomethylation.

P4-5 A study on cerebrospinal fluid biomarkers for fronto-temporal lobar degeneration 前頭側頭葉変性症の脳脊髄液バイオマーカーの研究 Tsuchimoto Yoko（土本瑶子）^1,2，服部功太郎^2,3，秀瀬　真輔²，篠山　大明²，宮川友子³，蔵下智子³，塚本忠⁴，水澤英洋⁵，功刀浩² ¹Biotech. and Life Sci., Tokyo Univ. of Agr and Tech. ²Department of Mental Disorder Research, National Institute of Neuroscience, NCNP, Japan ³Medical Genome Center, NCNP, Japan ⁴National Center of Neurology and Psychiatry hospital, NCNP, Japan ⁵NCNP Objectives: Fronto-temporal lobar degeneration (FTLD) is a subtype of dementia affecting the frontal and temporal lobes, presents various behavioral and language functional deficits. Recent brain image studies suggest the iron deposition in the brains of FTLD patients. Cerebrospinal fluid (CSF) is the biological fluid in closest contact with the brain and is particularly useful in the search of biomarkers of brain diseases. We aimed to find CSF biomarkers for FTLD, focusing on iron-related molecules.Methods: CSF samples from 17 patients with FTLD (age 67.5 ± 6.9 years) and 17 healthy controls (70.5 ± 10.9) were collected at the National Center of Neurology and Psychiatry (NCNP) Hospital after obtaining written informed consent and stored at the NCNP biobank. The levels of ferritin, transferrin and 14-3-3 protein were analyzed using Enzyme-linked immune assay (ELISA).Results: The mean CSF ferritin level in the FTLD group was similar to that of the control group. The mean transferrin level was lower in the FTLD group than in the control group, although the difference did not reach the statistical significance (p=0.126, ANCOVA, adjusted for age and sex). When male and females were analyzed separately, the mean transferrin level in male patients tended to be lower than that of male controls (p=0.052, ANCOVA). The mean 14-3-3 protein level in the FTLD group was significantly higher than in the control group (p=0.015, ANCOVA).Conclusion: The results raises the possibility that iron transport is altered in male FTLD patients. The observed increase in levels of 14-3-3 protein, which participates in monoamine transmission, could be underlie the pathophysiology of this disorder. Future studies using a larger sample size together with thorough clinical evaluation are warranted.		P4-6 Role of microglia in astrogliopahty: a study of Alexander disease model アレキサンダー病モデルにおけるミクログリアの役割 Shigetomi Eiji（繁冨英治）¹，小林憲司¹，齋藤光象^1,2，小泉　修一¹ ¹Dept. Neuropharmacol., Interdiscipl. Grad. Sch. of Med., Univ. Yamanashi, Yamanashi, Japan ²Dept Neurol, Grad. Sch. of Med. Sci., Kyoto Prefect. Univ. of Med. Alexander disease (AxD) is a rare neurodegenerative disease which is caused by dominant gain of function mutation for GFAP. Astrocytes in AxD display reactive phenotype of astrocytes which is characterized by Rosenthal fibers. Although AxD is thought be an astrogliopathy, emerging evidence suggest that AxD astrocytes cause a pronounced inflammatory response including microglial activation. However, the role of such inflammatory response in AxD pathogenesis remains elusive. In hemizygotes of 60TM mutant mice which harbor R239H mutation of human GFAP, we observed increase in Iba1-positive cells in the hippocampus at P21 and P42, in parallel with GFAP upregulation and accumulation. The number of Iba1-positive signals were positively correlated with GFAP-positive signals. Iba1-positive microglia showed ramified morphology without marked morphological changes. In order to understand the role of microglial responses in 60TM, we depleted microglia by treatment of CSF-1 receptor (CSF-1R) antagonist between P21 and P42. Iba1-positive signals were dramatically reduced and rarely seen, suggesting that microglia almost disappeared. Surprisingly, GFAP expression was augmented in 60TM mice with CSF-1R antagonist. Rosenthal fibers labeled by Fluoro-Jade B also increased with CSF-1R antagonist. It has been suggested that GFAP expression level is positively co-related with the disease progression of AxD. Thus, these data suggest that microglia in AxD model at early-developmental stage play a beneficial role in AxD pathogenesis.

P4-7 Screening of familial ALS/FTLD causative genes identifies mechanism of TDP-43 aggregation in cultured neuronal cells 家族性ALS/FTLD 原因遺伝子のスクリーニングによるTDP-43 凝集メカニズムの探索 Inami Hidekazu（稲見英和），渡邊征爾，山中宏二 Dept. Neurosci. Pathobiol., RIEM, Nagoya Univ. Mislocalization and aggregation of TAR DNA binding protein-43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Although over 25 genes were identified as causative genes of inherited ALS/FTLD, the precise mechanism(s) of intracellular aggregation of TDP-43 was not fully elucidated. In this study, we aimed to identify genes directly involved in TDP-43 aggregation. Using neuro-2a cells stably expressing GFP fused TDP-43 mutants lacking nuclear localization signal (N2a･TDP(dNLS)-EGFP) and a mammalian expression library of the ALS/FTLD causative genes, we classified the ALS/FTLD causative genes into three groups: 1. Genes co-aggregate with TDP-43 (PFN1, FUS etc.), 2. Genes form aggregates without TDP-43 (VAPB, SQSTM1 etc.), 3. Genes do not aggregate (TBK1, hnRNPA1 etc.). The group 1 gene products, aggregating with TDP-43, also induced mislocalization and aggregation of wild-type TDP-43 in neuro-2a cells. We found that most of the group 1 gene products were associated with microtubules such as PFN1 and TUBA4A, suggesting that microtubular dysfunction might be deeply involved in TDP-43 aggregation. Moreover, we also found that RNA binding proteins, including TDP-43 itself, were highly co-aggregated with TDP-43 when they are leaked from nuclei. This result suggests that preventing cytosolic leakage of RNA binding proteins may be a potential therapeutic target for ALS/FTLD.		P4-8 Identification of MAM disruption as a general pathological mechanism in ALS ALS共通病態としての小胞体・ミトコンドリア膜間領域（MAM）破綻の検証 Watanabe Seiji（渡邊征爾），山中宏二 Dept. Neurosci. Pathobiol., RIEM, Nagoya Univ. Mitochondria-associated membrane (MAM) is a contact site of mitochondria and endoplasmic reticulum (ER). MAM is important for intracellular homeostasis such as calcium supply to mitochondria, ER stress response, and autophagy. We previously reported that collapse of MAM is a common pathological mechanism in SOD1- and SIGMAR1-linked amyotrophic lateral sclerosis (ALS). To address the question whether the MAM disruption is a fundamental pathomechanism in ALS, we first tried to visualize the MAM disruption in living cells using a novel fluorescent reporter, pMAM-tracker. pMAM-tracker is a plasmid coding ddGFP, a dimerization-dependent green fluorescent protein (Alford et al. (2012) ACS Synth Biol). To enable polycistoronic expression of a mitochondria or ER targeted ddGFP, a sequence coding 2A peptide was also included into pMAM-tracker. As expected, ddGFPs was localized to mitochondria and ER, including MAM, in neuro2a and HeLa cells. Fluorescent intensity was decreased by expression of mutant SOD1 protein or suppressing SIGMAR1 expression, which are known to induce MAM disruption. Moreover, we established cell lines stably expressing pMAM-tracker as reporter lines of MAM disruption. With the reporter cell lines and a mammalian expression library of ALS causative genes, we are in process to examine whether the various ALS causative genes generally induce MAM disruption as similar to SOD1 and SIGMAR1. Our product will contribute not only to understanding the pathological mechanism of ALS, but also to examining the role of MAM in other neurodegenerative diseases.