パーキンソン病とその類縁疾患 Parkinson's Disease and Related Disorders
P3-1-207 マカクザルにおけるアルファシヌクレイン発現ニューロンの加齢による分布変化 Age-dependent changes in the distribution of alpha-synuclein-expressing neurons in macaque monkeys ○木村活生1,2, 井上謙一1, 黒田呈子1, 田中章景2, 高田昌彦1 ○Katsuo Kimura1,2, Ken-ichi Inoue1, Teiko Kuroda1, Fumiaki Tanaka2, Masahiko Takada1 京都大・霊長研・統合脳システム1, 横浜市大院・医・神経内科学2 Div Sys Neurosci, Pri Res Isnt, Kyoto Univ, Inuyama, Japan1, Dept Neurol, Grad Sch Med, Yokohama City Univ, Yokohama, Japan2 Parkinson's disease (PD) is a progressive neurodegenerative disease that is characterized by selective loss of dopamine neurons from the substantia nigra. An interaction between dopamine and alpha-synuclein (α-syn) leads to the dopamine neuron death and the inclusion of misfolded α-syn in intracellular Lewy bodies. According to the Braak's classification known as a model of the progression of PD pathology, α-syn deposition initially appears in the dorsal IX/X motor nucleus and/or the olfactory bulb and spreads to various regions of the brain. Although the α-syn deposition is also found during normal aging, it remains unclear how the deposition proceeds and spreads. In the present study, we investigated the distribution pattern of α-syn-expressing neurons in aged macaque brains. Six macaque monkeys (two cynomolgus monkeys and four Japanese monkeys) ranging 10-31 years old were used. We performed counts of nigral neurons immunoreactive to α-syn and tyrosine hydroxylase (TH). As the age went, the number of α-syn-positive neurons was increased, while that of TH-positive neurons was decreased. Moreover, loss of TH-positive neurons was observed more drastically in the ventral tier than in the dorsal tier of the nigra where calbindin is coexpressed in dopamine neurons. At younger ages (10-14 years old), α-syn-expressing neurons were located not only in the nigra, but also in the amygdala and hippocampus. At older ages (25-31 years old), they further appeared in frontal cortical areas (such as the cingulate and orbital regions) and the diagonal band of Broca and/or the basal nucleus of Mynert. In these aged monkeys, the number of nigral neurons expressing α-syn or TH was 170-265% or 51-72% as many as in the 10-year-old case, respectively. However, no conspicuous motor deficits were observed even in the aged monkeys. The present results suggest that aged macaque monkeys are useful as a potential presymptomatic model of PD.	P3-1-208 グルコセレブロシダーゼを完全に欠損したメダカは脳にアルファシヌクレイン凝集体を形成する Viable Gaucher's disease model of medaka fish completely deficienct in glucocerebrosidase activity developed alpha-synuclein aggregation in the brain ○上村紀仁1, 松井秀彰5, 藤原 石川智子2, 木下政人3, 小池正人6, 山門穂高1, 植村健吾1, 内山安男1, 武田俊一4, 藤堂剛2, 高橋良輔1 ○Norihito Uemura1, Hideaki Matsui5, Tomoko Fujiwara-Ishikawa2, Masato Kinoshita3, Masato Koike6, Hodaka Yamakado1, Kengo Uemura1, Yasuo Uchiyama1, Shun-ichi Takeda4, Takeshi Todo2, Ryosuke Takahashi1 京都大学大学院医学研究科臨床神経学1, 大阪大学大学院医学系研究科放射線基礎医学2, 京都大学大学院農学研究科応用生物科学専攻海洋生物機能学分野3, 京都大学大学院医学研究科放射線遺伝学4, 順天堂大学大学院医学研究科神経機能構造学6 Department of Neurology, Kyoto University Graduate School of Medicine, Kyoto, Japan1, Department of Radiation Biology and Medical Genetics, Osaka University Graduate School of Medicine, Suita, Japan2, Division of Applied Biosciences, Kyoto University Graduate School of Agriculture, Kyoto, Japan3, Department of Radiation Genetics, Kyoto University Graduate School of Medicine, Kyoto, Japan4, Technische Universitat Braunschweig, Germany5, Department of Cell Biology and Neuroscience, Juntendo University Graduate School of Medicine, Tokyo, Japan6 Objective: To clarify the pathogeneticl mechanism underlying glucocerebrosidase (GBA) mutations leading to Parkinson's disease (PD).Background: GBA is a causative gene of Gaucher's disease (GD), a lysosomal storage disease. Heterozygous mutations in this gene have been identified as the most common and important risk factor for PD. However, the underlying mechanisms remain unclear.Methods: We generated GBA mutant medaka by screening a TILLING (Targeting Induced Local Lesions In Genomes) library and analyzed their phenotype.Results: 1 nonsense mutation and 10 missense mutations were found in the TILLING library, and a nonsense mutant medaka and 2 missense mutant medaka were generated by in vitro fertilization. The homozygous nonsense mutants showed no glucocerebrosidase enzymatic activity. These mutants showed abnormal swimming movement at 2 months and died by 5 months. Immunohistochemical analysis revealed alpha-synuclein aggregation in the brain and histopathological features similar to that of neuronopathic GD. These phenotypes were rescued by the transgenic expression of GBA driven by growth-associated protein 43 (GAP-43) promoter.Conclusions: The homozygous nonsense mutant medaka survived for months in contrast to GBA knockout mice, which died soon after birth. These mutants showed pathologies related to alpha-synuclein as well as neuronopathic Gaucher's disease. These results indicate that GBA mutant medaka could be an excellent animal model for analyzing the molecular association between GBA mutations and alpha-synuclein accumulation, leading to PD.
P3-1-209 マウスの脳における核酸の酸化損傷防御酵素群、MTH1, OGG1, MUTYHの発現 Expression of the defense enzymes MTH1, OGG1 and MUTYH against oxidative damage in nucleic acids in the mouse brain ○盛子敬1,2, 中別府雄作1,2 ○Zijing Sheng1,2, Yusaku Nakabeppu1,2 九州大学・生体防御医学研究所　個体機能制御学部門　脳機能制御学分野1, 九州大学・ヌクレオチドプール研究センター2 Division of Neurofunctional Genomics, Department of Immunobiology and Neuroscience, Medical Institute of Bioregulation,Fukuoka, JAPAN1, Research Center for Nucleotide Pool, Kyushu University, JAPAN2 Oxidative DNA damage has been inferred to be involved in the neurodegenerative diseases as well as brain aging. To counteract oxidative damage to nucleic acids, human and rodents are equipped with three defense enzymes, MTH1, OGG1 and MUTYH. MTH1 hydrolyzes oxidized purine nucleoside triphosphates to the monophosphates, thus sanitizing nucleotide pools. OGG1, an 8-oxoguanine DNA glycosylase, prevents buildup of 8-oxoguanine in both nuclear and mitochondrial genomes. MUTYH, an adenine DNA glycosylase, excises adenine opposite 8-oxoguanine and 2-hydroxyadenine opposite guanine. Using knockout mice of each gene, we have shown that MTH1, OGG1, or MUTYH play different roles in various brain regions such as hippocampus, striatum or substantia nigra under increased oxidative stress (Sheng et al., J Clin Invest, 2012; Yamaguchi et al., Cell Death Differ, 2006). However, their expression and distribution in the mouse brain have yet to be inestigated in detail. In the present study, the expression of MTH1, OGG1 and MUTYH proteins in the mouse brain was examined immunohistochemically with light microscopy (LM), laser scanning confocal microscopy (LSCM) and electron microscopy (EM) . First, we observed that expression of MTH1, OGG1 or MUTYH is abundantly detected in various brain regions, including the striatum, hippocampus, and substantia nigra. LSCM confirmed that various neuronal marker-positive cells express MTH1, OGG1 or MUTYH, respectively. Furthermore, EM revealed that immunogold particles for MTH1, OGG1 or MUTYH, are detected in nuclei, cytoplasm, and mitochondria of neurons, respectively. These results strongly suggest that these defense enzymes contribute to the maintenance of nuclear and mitochondrial genomic integrities in neurons, thus may play important roles in protection of brains from oxidative stress. Our results provide important morphological bases for further understanding of functions of these enzymes in the mouse brain.	P3-1-210 ATP13A2変異メダカの解析 Analysis of ATP13A2 mutant medaka ○樽野陽亮1, 上村紀仁1, 松井秀彰1, 山門穂高1, 高橋良輔1 ○Yosuke Taruno1, Norihito Uemura1, Hideaki Matsui1, Hodaka Yamakado1, Ryosuke Takahashi1 京都大学大学院　医学研究科　脳病態生理学講座　臨床神経学1 Department of Neurology, Kyoto university graduate school of medicine, Kyoto, Japan.1 <Introduction>PARK9, reported as Kufor-Rakeb syndrome by al-Din et al. in 1994, is the autosomal recessive juvenile onset familial Parkinson's disease, and characterized by L-DOPA responsible parkinsonism, cognitive dysfunction, pyramidal signs and myoclonus. The gene responsible for PARK9 was recently identified as ATP13A2, and ATP13A2 protein was found to have 10 transmembrane domains and locate in the lysosome as P-type ATPase encoded 1180 amino acid. The function of ATP13A2 protein was unknown. It is assumed that loss of the lysosomal function as well as gain of toxic function by mutant ATP13A2 were responsible for the neuronal loss in PARK9, although the precise mechanism of it is not well understood.<Methods>To reveal the mechanism of neuronal loss in PARK9, we created ATP13A2 mutant medaka by TILLING (Tilling is Targetting Induced Local Lesions In Genome) method using ENU mutagen.<Results and conclusions>The point mutation in the ATP13A2 mutant medaka was located in the intron between exon 12 and 13 (IVS13, T-C, +2), and causes exon 13 skipping in the ATP13A2 gene transcription, which is relevant to human PARK9 cases. Aged ATP13A2 mutant medaka showed reduced dopamine content, and the number of TH+ neurons in the midbrain was also decreased, suggesting that ATP13A2 mutant medaka could be a good PARK9 model. Although the α-synuclein accumulation by ATP13A2 knockdown in mice primary cortical neurons was reported, it is still unknown in vivo. Now we are performing immunohistochemical analysis including α-synuclein immnostaining in ATP13A2 mutation medaka to confirm the relationship between the dysfunction of ATP13A2 and α-synuclein accumulation.
P3-1-211 パーキンソン病モデルマウスにおけるヒト間葉系および造血幹細胞の移植効果 Therapeutic effects of human mesenchymal and hematopoietic stem cells in parkinsonian mice ○北村佳久1, 位田雅俊2, 高田和幸1, 芦原英司1, 下濱俊3 ○Yoshihisa Kitamura1, Masatoshi Inden2, Kazuyuki Takata1, Eishi Ashihara1, Shun Shimohama3 京都薬大・病態生理1, 岐阜薬大・薬・薬物治療2, 札幌医大・医・神経内科3 Dept. Clin. Transl. Physiol., Kyoto Pharm. Univ., Kyoto1, Lab. Med. Therap. Mol. Therap., Gifu Pharm. Univ., Gifu2, Dept. Neurol., Sapporo Med. Univ., Sapporo3 To appreciate the potential applications of stem cell technology to neurodegenerative diseases including Parkinson's disease (PD), it is important to understand the characteristics of the various stem cell types available. In this study, we designed a set of experiments to compare the ability of 3 human adult stem cells - mesenchymal stem cells (MSCs), bone marrow CD34+ cells (BM), cord blood CD34+ cells (CB) - using rotenone-treated NOD/SCID mice. Rotenone was administered orally once a day at doses of 30 mg/kg for 56 days to induce a Parkinsonian phenotype. Intravenous delivery of CB into rotenone mice was slightly more efficient for benefits that were confirmed by both histological and behavioral analyses than that of MSCs or BM. A few human nuclei (hNu)+ cells, a specific maker of human cells, only in the striatum were observed in rotenone-treated mice transplanted with stem cells. These hNu+ cells expressed tyrosine hydroxylase (TH), a maker of dopamine neurons. Additionally, α -synuclein+/TH+ cells in the substantia nigra pars compacta were significantly decreased by injection of stem cells into the tail vein. Gene expressing analysis using RT-PCR also revealed that MSCs, BM and also CB expressed some neurotrophic factors including NGF, BDNF, etc. These results suggest that the benefical effects of intravenous delivery of stem cells into rotenone mice may result not only from a neurotrophic effect but also from endogenous brain repair mechanisms. These results also demonstrate that the potentials of intravenous delivery of stem cells derived from an autologous source for clinical applications of PD.	P3-1-212 生体液中のα-シヌクレインは単量体で存在する α-Synuclein is present as a monomer in the biological fluid ○建部陽嗣1, 徳田隆彦1,2, 石井亮太郎1, 渡邊義久3, 田口勝敏3, 笠井高士1, 辻村敦3, 水田依久子1, 水野敏樹1, 田中雅樹2, 中川正法1 ○Harutsugu Tatebe1, Takahiko Tokuda1,2, Ryotaro Ishi1, Yoshihisa Watanabe3, Katsutoshi Taguchi3, Takashi Kasai1, Atsushi Tsujimura3, Ikuko Mizuta1, Toshiki Mizuno1, Masaki Tanaka2, Masanori Nakagawa1 京都府立医科大学　神経内科学1, 京都府立医科大学　分子脳病態解析学2, 京都府立医科大学　基礎老化学3 Department of Neurology, Kyoto Prefectural University of Medicine, Kyoto1, Department of Molecular Pathobiology of Brain Disease, Kyoto Prefectural University of Medicine, Kyoto2, Department of Basis Geriatrics, Kyoto Prefectural University of Medicine, Kyoto3 Some recent studies suggested that α-synuclein (α-syn) (molecular weight: 14 kDa) exists predominantly as a tetramer (~58 kDa) in the fluid. However, there is a subsequent report showing the brain- and RBC-derived α-syn protein is present as a natively unfolded monomer, and what the native structure of α-syn is therefore still controversial. To determine a higher molecular structure of the α-syn protein in the biological fluid, we analyzed cerebrospinal fluid (CSF) or red blood cells fractionated by size-exclusion chromatography (SEC) with a Superose12 column as well as Western blot (WB), ELISA, and MALDI/TOF MS. We also expressed A140C α-syn variant that formed α-syn dimers and wild-type α-syn in Escherichia coli (recombinant α-syn proteins), and then analyzed them after fractionation by the SEC. We compared recombinant α-syn proteins with native α-syn in the biological fluids. We confirmed that α-syn in the biological fluid was eluted in an about 60kDa fraction in the SEC, but a protein band was detected at ~14 kDa as a monomer in SDS-PAGE and WB. The similar result was obtained in recombinant wild-type α-syn. We also confirmed by MALDI/TOF MS that molecular weight of CSF α-syn eluted in the 60kDa fraction in the SEC was ~14 kDa. On the other hand, A140C α-syn variant was eluted in the fractions of 100kDa or more in the SEC, and α-syn band was detected at ~28kDa in SDS-PAGE and WB. These results suggest that α-syn detected in about 60kDa fraction in the SEC was a monomeric protein, implying that α-syn in the biological fluid exists as a monomer, not a tetramer.
P3-1-213 ドーパミンシグナルはマウス線条体サイクリン依存性キナーゼ 5 (Cdk5) のチロシン 15 残基リン酸化を抑制的に制御する Dopamine signaling negatively regulates striatal phosphorylation of Cdk5 at tyrosine 15 in mice ○山村行生1,2, 森垣龍馬2,3, 笠原二郎1, 横山宏典1, 田辺彬恵1, 大北真哉2,3, 小泉英貴4, 永廣信治2,3, 梶龍兒2,4, 後藤恵2,3,4 ○Yukio Ymamura1,2, Ryoma Morigaki2,3, Jiro Kasahara1, Hironori Yokoyama1, Akie Tanabe1, Sinya Okita2,3, Hidetaka Koizumi4, Shinji Ngahiro2,3, Ryuji Kaji2,4, Satoshi Goto2,3,4 徳島大院・ヘルスバイオサイエンス（薬）・神経病態解析1, 徳島大・パーキンソン病・ジストニア研究センター2, 徳島大院・ヘルスバイオサイエンス（医）・脳神経外科3, 徳島大院・ヘルスバイオサイエンス（医）・臨床神経科学4 Dept Neurobiol Theapeu, Grad Sch Pharmaceu Sci, Inst Health Biosci, Univ Tokushima, Tokushima, Japan1, Parkinson's Disease and Dystonia Research Center, Univ Tokushima, Tokushima, Japan2, Dept Neurosurgery, Grad Sch Medic Sci, Inst Health Biosci, Univ Tokushima, Tokushima, Japan3, Dept Clinic Neurosci, Grad Sch Medic Sci, Inst Health Biosci, Univ Tokushima, Tokushima, Japan4 Striatal functions depend on the activity balance between the dopamine (DA) and glutamate (Glu) neurotransmissions that produce opposing physiological effects. Glu inputs activate cyclin-dependent kinase 5 (Cdk5), which inhibits postsynaptic DA signaling by phosphorylating DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, 32 kDa) at Thr75 (Thr75-DARPP-32) in the striatum. DARPP-32 integrates the activities of dopaminergic and glutamatergic transmissions, and it is therefore thought to play as a key regulator for striatal activities. c-Abelson tyrosine kinase (c-Abl) is known to phosphorylate Cdk5 at Tyr15 (Tyr15-Cdk5) and thereby facilitates the Cdk5 activity. We here report that Cdk5 with Tyr15 phosphorylation (Cdk5-pTyr15) is enriched in the mouse striatum, where dopaminergic stimulation inhibited phosphorylation of Tyr15-Cdk5 by acting through the D2 class DA receptors. Moreover, in the 1-methyl-4-phenyl-1,2,4,6-tetrahydropyridine (MPTP) mouse model, DA deficiency caused increased phosphorylation of both Tyr15-Cdk5 and Thr75-DARPP-32 in the striatum, which could be attenuated by administration of L-dopa or imatinib (STI-571), a selective c-Abl inhibitor. Our results suggest a functional link of Cdk5-pTyr15 with postsynaptic DA and Glu signals through the c-Abl kinase activity in the striatum, and possibility of these molecules as novel targets for PD therapeutics.	P3-1-214 ロテノン神経毒性に対する中枢および末梢神経系のニューロンとグリアの変化とメタロチオネインの関与 Effect of systemic rotenone exposure on central and peripheral nerveous systems and involvement of metallothionein ○村上真樹1, 宮崎育子1, 十川紀夫2, 浅沼幹人1 ○Shinki Murakami1, Ikuko Miyazaki1, Norio Sogawa2, Masato Asanuma1 岡山大学大学院　医歯薬学総合研究科　神経情報学1, 歯科薬理学2 Dept. of Brain Sci., Okayama Univ. Grad. Sch. of Med., Dent. & Pharamceut. Sci., Okayama.1, Dept. of Dental Pharmacol.2 Parkinson's disease (PD) is a neurodegenerative disease with motor symptoms as well as non-motor symptoms that precede the onset of motor symptoms. Mitochondrial complex 1 inhibitor, rotenone has been widely used to reproduce pathology in central nervous system (CNS) and peripheral nervous system (PNS). We have demonstrated that chronic systemic rotenone exposure caused alteration of rat enteric neuronal and glial cells, which precedes neurodegeneration in CNS. While, we previously reported that released metallothioneins (MTs) from astrocytes can protect dopaminergic neurons against oxidative stress. Here, we investigated the effects of the chronic systemic rotenone exposure on CNS and PNS of MT-1, -2 knock out (KO) mice. Rotenone administration caused reduction of dopamine nerve terminal accompanying progressively decreased spontaneous motor activity in MT KO mice, compared with that in WT mice. Furthermore, the chronic administration of rotenone reduced various functional proteins in neuronal and glial cells of colon and epicardial nerve fascicle in MT KO mice. The present results suggest that the susceptibility of PNS to rotenone is different from that of CNS, and MT also act protective against rotenone-induced neuronal damage in PNS.
P3-1-215 ゾニサミド、レボドパ併用によるパーキンソン病モデルラット線条体での遺伝子発現への影響 Effect of zonisamide co-administration with levodopa on global gene expression in the striata of rats with Parkinson's disease ○小島俊男1,2, 渡嘉敷尚子3, 金丸愛3, 植田勇人3 ○Toshio Kojima1,2, Shoko Tokashiki3, Ai Kanemaru3, Yuto Ueda3 浜松医大・機器センター1, 豊橋技科大・体育保健センター2, 宮崎大学・医・精神医学3 Res Equip Ctr, Hamamatsu Univ Sch Med, Hamamatsu1, Res Ctr Phys Fitn Sports Health, Toyohashi Univ Tech, Toyohashi2, Sect Psychiat, Univ Miyazaki, Miyazaki3 The anti-epileptic drug zonisamide is reported to exert beneficial effects in patients with Parkinson's disease. To elucidate the pathophysiological mechanisms underlying the anti-parkinsonism effects of zonisamide, we examined the effect of zonisamide co-administered with levodopa in the striata of rats with 6-hydoroxydopamine hemiparkinsonism by using a DNA microarray for genome-wide gene expression profiling. We found that the expression of some genes related to metabolism and nervous system development and function were upregulated by zonisamide; expression of these genes was downregulated by levodopa. Furthermore, many genes related to the immune system and inflammation were downregulated by zonisamide, and their expression was upregulated by levodopa. These results indicate that zonisamide has a protective effect when co-administered with levodopa.	P3-1-216 視覚誘導性手首運動における3つの運動成分の同定と神経疾患の病態分析への応用 Identification of three components of tracking movement of the wrist joint and its application for analysis of neurological disorders ○李鍾昊1, 織茂智之2, 筧慎治1 ○Jongho Lee1, Satoshi Orimo2, Shinji Kakei1 東京都医学総合研究所・運動失調プロジェクト1, 関東中央病院・神経内科2 Motor disorders project, Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan1, Department of Neurology, Kanto Central Hospital, Tokyo, Japan2 Recently, we developed a novel method to dissociate motor commands for visually guided smooth pursuit movement of the wrist joint into two components from two parallel controllers. One component that belonged to the lower frequency range (=F1 domain, 0~0.5Hz) represents an output from a predictive controller that synchronizes movement of the wrist joint with motion of the target continuously. The other component that belonged to the higher frequency range (=F2 domain, 0.5~3Hz) represents an output from a feedback controller that corrects positional errors intermittently. In the present study, we identified the third component of the wrist movement that belonged to even higher frequency range (=F3 domain, 4~8Hz), and compared the smooth pursuit movements of normal controls, Parkinson diseases and cerebellar patients based on the three control modes. The smooth pursuit movement of the normal controls was performed predominantly by the predictive controller and supported by the feedback controller. In contrast, in case of the cerebellar patients, the more the severity of the illness, the less the accuracy and contribution of the predictive controller. The malfunction of the predictive controller in these patients was supplemented by a number of step-wise corrective movements from the feedback controller. Consequently, the movement kinematics of the cerebellar patients was characterized by the lack of smoothness. In normal and cerebellar patients, the third, F3 component (4~8Hz) was minor if any. On the other hand, the wrist tracking movement of patients with Parkinson's disease showed marked increase in the movement component in F3 domain.Overall, our method provides a new quantitative tool to make functional evaluation of neurological disorders such as Parkinson's disease or cerebellar ataxia in terms of properties of motor controllers.
P3-1-217 加齢バエおよびパーキンソン病モデルショウジョウバエの睡眠覚醒リズム異常の解析 Analysis of sleep-wake rhythms in aged and Parkinson's disease model flies ○梅崎勇次郎1,2, 吉井大志2,3富岡憲治2, 服部信孝4,5, 今居譲4,5 ○Yujiro Umezaki1,2, Taishi Yoshii2,3, Charlotte Helfrich-Förster3, Kenji Tomioka2, Nobutaka Hattori4,5, Yuzuru Imai4,5 順天堂大学大学院医学研究科　老人性疾患病態・治療センター1, 岡山大学大学院自然科学研究科2, 順天堂大学神経変性疾患病態治療探索講座4, 順天堂大学脳神経内科5 Research Institute for Diseases of Old Age, Juntendo University Graduate School of Medicine, Tokyo, Japan1, Graduate School of Natural Science and Technology, Okayama University, Okayama, Japan2, Department of Neurobiology and Genetics, Biocenter, University of Würzburg, Würzburg, Germany3, Department of Neuroscience for Neurodegenerative Disorders, Juntendo University Graduate School of Medicine, Tokyo, Japan4, Department of Neurology, Juntendo University Graduate School of Medicine, Tokyo, Japan5 Rest-activity rhythms driven by an endogenous control mechanism are associated with sleep activity in Drosophila, which is impaired with advancing age. However, the molecular mechanism underlying age-dependent disturbance in sleep is largely unknown. We investigated rest-activity rhythms under light-dark cycles (LD) and constant darkness (DD) in young (1-day-old) and middle-aged (30- and 40-day-old) male wild-type flies. Middle-aged flies showed reduced daily total activity in LD and the significantly lengthened the free-running period by approximately 1 hr in DD. We then compared pigment-dispersing factor (PDF)-immunoreactivity in two groups of clock neurons, i.e. s-LNv and l-LNv, among young (5-day-old), middle aged (40- and 50-day-old) and old (65-day-old) flies. The immunoreactivity in axons of l-LNv was decreased with age, and the area of PDF-positive axonal terminals of s-LNv was also reduced. These results suggest that reduction of PDF levels is involved in age-dependent rhythm changes. Parkinson's disease (PD) is one of age-associated neurodegenerative diseases and patients with PD often show disturbance in sleep. We also examined sleep-wake cycles of flies harboring mutant PD genes, α-synuclein and leucine rich-repeat kinase 2 (LRRK2). Both of PD model flies showed reduced sleep levels for the first half of nighttime, suggesting that pathogenic mutations of α-synuclein and LRRK2 affect, at least in part, sleep behavior. Our study provides the possibility that Drosophila is a useful mode for sleep disturbance in aging and PD pathogenesis.	P3-1-218 パーキンソン病患者 iPS 細胞由来神経細胞における酸化ストレス応答ならびに代謝動態の解析 Analyses of oxidative stress and metabolic dynamics in Parkinson's disease specific iPS cells-derived neurons ○葛巻直子1, 今泉陽一1, 赤松和土1, 岡田洋平1,2, 伊澤奈々1,3,4, 松本拓也1, 菱木貴子5, 長畑善子5, 須田雪明6, 成田年6, 末松誠5, 服部信孝3, 岡野栄之1 ○Naoko Kuzumaki1, Yoichi Imaizumi1, Wado Akamatsu1, Yohei Okada1,2, Nana Izawa1,3,4, Takuya Matsumoto1, Takako Hishiki5, Yoshiko Nagahata5, Yukari Suda6, Minoru Narita6, Makoto Suematsu5, Nobutaka Hattori33, Hideyuki Okano1 慶應義塾大学医学部　生理学1, 慶應義塾大学医学部　咸臨丸プロジェクト2, 順天堂大学医学部　脳神経内科3, 順天堂大学医学部　リハビリテーション医学4, 慶應義塾大学医学部　医化学5, 星薬科大学　薬理学教室6 Dept. Physiol., Keio Univ. Sch. Med. Tokyo1, Kanrinmaru Prj., Keio Univ. Sch. Med. Tokyo2, Dept. Neurol., Juntendo Univ. Sch. Med., Tokyo3, Dept. Reha., Med., Juntendo Univ. Sch. Med., Tokyo4, Dept. Biochem., Keio Univ. Sch. Med. Tokyo5, Dept. Pharmacol., Hoshi Univ. Sch. Pharm. Pharmaceut. Sci., Tokyo6 In the present study, we evaluated the change in the oxidative stress and energy metabolism of familial Parkinson's disease specific induced pluripotent stem cells (iPSCs)-derived neurons. iPSCs were generated from dermal fibroblasts isolated from two PARK2 patients carrying parkin mutations and two control subjects. All of the clones differentiated into neurons were including tyrosine hydroxylase-positive neurons. Neurons-derived from PARK2 iPSCs, but not fibroblasts or iPSCs, exhibited abnormal mitochondrial morphology and impaired mitochondrial homeostasis. Under these conditions, we examined oxidative metabolism in the iPSCs derived neurons by measuring the cellular levels of reduced glutathione (GSH). GSH reacts with reactive oxygen species (ROS) and is catalyzed by glutathione S-transferase. The levels of GSH in PARK2 iPSC-derived neurospheres were significantly lower as compared to that in control iPSC-derived neurospheres. It has been reported that the Nrf2 pathway plays a cytoprotective role under conditions of ROS accumulation. The expression of Nrf2 pathway proteins, such as Nrf2 and NADH quinone oxidoreductase (NQO1), was significantly increased in PARK2 iPSCs-derived neurons, indicating an increased level of oxidative stress accompanied by activation of the Nrf2 pathway in PARK2 neurons. Whereas, Nrf2 has been shown to enhance several metabolic gene expressions in cancer cells. Following electrophoresis mass spectrometry (CE-MS) system, we found that parkin deficiency activated glycolysis and reduced mitochondrial respiration in neurons-derived from PARK2 iPSCs. These findings suggest that, although further studies are still needed, PARK2 iPSCs-derived neurons may affect oxidative stress and abnormal metabolism associated with the dysfunction of mitochondrial homeostasis.
P3-1-219 alpha-synucleinのメチオニン127におけるドーパミン依存的な酸化修飾は細胞毒死と重合化を引き起こす Dopamine-mediated oxidation of methionine 127 in alpha-synuclein causes cytotoxicity and oligomerization of alpha-synuclein ○中曽一裕1, 田島奈緒子1, 伊藤悟2, 堀越洋輔1, 松浦達也1 ○Kazuhiro Nakaso1, Naoko Tajima1, Satoru Ito2, Yosuke Horikoshi1, Tatsuya Matsuura1 鳥取大学医学部　病態解析医学講座　統合分子医化学分野1, 鳥取大学　医学部　脳神経内科2 Div. Med Biochem, Fac of Med. Tottori Univ.1, Dep Neurology, Fac of Med. Tottori Univ.2 Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and the presence of Lewy bodies. Many recent studies focused on the interaction between alpha-synuclein (a-syn) and dopamine in the pathogenesis of PD. However, it is not well understood why PD-related pathogenesis occurs selectively in dopaminergic neurons. We investigated the interaction between dopamine and a-syn with regard to cytotoxicity. A soluble oligomer was formed by co-incubating a-syn and dopamine in vitro. To clarify the effect of dopamine on a-syn in cells, we generated PC12 cells expressing human a-syn, as well as the a-syn mutants, M116A, Y125D, M127A, S129A, and M116A/M127A, in a tetracycline-inducible manner (PC12-TetOFF-a-syn). Overexpression of wildtype a-syn in catecholaminergic PC12 cells decreased cell viability in long-term cultures, while a competitive inhibitor of tyrosine hydroxylase blocked this vulnerability, suggesting that a-syn-related cytotoxicity is associated with dopamine metabolism. The vulnerabilities of all mutant cell lines were lower than that of wildtype a-syn-expressing cells. Moreover, a-syn containing dopamine-mediated oxidized methionine (Met(O)) was detected in PC12-TetOFF-a-syn. Met(O) was lower in methionine mutant cells, especially in the M127A or M116A/M127A mutants, but also in the Y125D and S129A mutants. Co-incubation of dopamine and the 125YEMPS129 peptide enhanced the production of H2O2, which may oxidize methionine residues and convert them to Met(O). Y125- or S129-lacking peptides did not enhance the dopamine-related production of H2O2. Our results suggest that M127 is the major target for oxidative modification by dopamine, and that Y125 and S129 may act as enhancers of this modification. These results may describe a mechanism of dopaminergic neuron-specific toxicity of a-syn in the pathogenesis of PD.	P3-1-220 Have CB2 receptor agonists neuroprotective effect in Parkinson's Disease? ○Asaadi Sina1, Zali Alireza1, Ashrafi Farzad1 Functional neurosurgery and stereotaxi research centre1 Introduction:Parkinson's disease (PD) is characterized by the progressive loss of nigrostriatal dopamine neurons. Current pharmacotherapies fail to prevent or slow down the disease progression. Although the ethiology of PD is presently unknown, major pathogenic processes, which trigger the progressive loss of nigral dopaminergic neurons, are oxidative stress, mitochondrial dysfunction, and inflammatory stimuli . In recent years CB1 receptors agonist have been proposed as clinically promising neuroprotective molecules in Parkinson Disease But there is less evidence that CB2 receptor agonists can have protective role in brain in response to neuronal damage during PD . in this study we investigated effect of CB2 receptor agonist ( HU-308) in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)induced PD model in male mice to assess the neuroprotective role of HU-308 in slowing down of PD progression. Materials and Methods: sixty mice were divided to three groups. One group were injected MPTP ( systemically) as control group. second group were injected MPTP and then HU-308, 7 day after injection of MPTP to assay preservation of dopamine function in brain. In third group after 2 day pretreatment with HU-308(intraperitoneal), MPTP were injected to assay neuroprotection effect of HU-308. Result:Behavioral assess showed significant improvement in pretreatment group with HU-308. Also Neurochemical analysis that were used to assess dopamine metabolites and immunohistochemistry studies after brain resection showed significant rise in dopamine metabolites , CB2 receptors upregulation and survival of dopaminergic neurons in compared with controls in brain. Conclusion: our data indicate that CB2 receptors agonist protects against MPTP-induced nigrostriatal degeneration by inhibiting microglial cells activation and suggest that CB2 receptors represent a new therapeutic target to slow the degenerative process occurring in PD.
P3-1-221 Decoding a limbic-motor network contribution to the expression of obsessive compulsive behavior in macaques ○Kevin W. McCairn1, Masaki Isoda2, Atsushi Iriki3, Masahiko Takada1, Masayuki Matsumoto1,4 Systems Neuroscience Section, Primate Research Institute, Kyoto University1, Department of Physiology, Kansai Medical University School of Medicine, Moriguchi, Osaka, Japan2, DIvision of Biomedical Science, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan3 Obsessive compulsive behavior (OCB) is a cardinal symptom of Tourette syndrome (TS), a complex hyperkinetic motor and neuropsychiatric disorder that is believed to arise from abnormalities within cortico-basal ganglia circuits (CBCs). Recent studies in a pharmacologically induced nonhuman primate model of TS, which utilizes microinjection of the GABA antagonist bicuculline into the motor territory of the striatum, have shown that the simple motor tics associated with TS are correlated with highly stereotyped neuronal activation patterns in the motor territories of the CBC. During tic events, neurons in the primary motor cortex (M1) enter a bi-stable state where the transition from the inter-tic interval to the tic state is associated with a phasic burst against a background of no activity, which drives muscle activation. The OCB symptoms of TS are believed to arise from abnormalities within the limbic territories of the CBC. Utilizing the same methodology as used for simple-tic induction, we targeted the limbic territory of the striatum to induce stereotypic behavior as an analogue of OCB. We found that during the transition to the OCB state, manifesting as repetitive chewing, the M1 and the anterior cingulate cortex (ACC) became strongly coherent in the low-frequency range &It5 Hz. A bi-stable state emerged in the ACC similar to previous observations of the M1 during simple motor tics. Single-cell activity in the M1, however, was characterized by a regular firing pattern that was punctuated by phasic activations. This pattern was strongly correlated with peak activity in the ACC and EMG activity. The present results demonstrate that complex stereotypic behavior emerges when low-frequency dysrhythmia becomes coherent between the limbic and motor networks.	P3-1-222 Withdrawn

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