ポリグルタミン病、ALS、脊髄小脳変性症、その他の神経変性疾患 Polyglutamine Diseases, ALS, SCD, Other Neurodegenerative Disorder
P1-1-215 変異スーパーオキシドジスムターゼ1（SOD1）はSOD1G93Aマウスにおいてミトコンドリア・小胞体膜間領域に蓄積する Mutant copper-zinc superoxide dismutase 1 (SOD1) accumulated at the mitochondria associated membrane in SOD1G93A transgenic mice model ○渡邊征爾1, 山中宏二1,2 ○Seiji Watanabe1, Koji Yamanaka1,2 理研・BSI・運動ニューロン変性1, 科技機構・CREST2 Lab. for Motor neuron diseases, RIKEN BSI1, CREST, JST2 A dominant mutation in the gene for copper-zinc superoxide dismutase 1 (SOD1) is the most frequent cause of the inherited form of amyotrophic lateral sclerosis (ALS). Mutant SOD1 provokes progressive degeneration of motor neurons by an unidentified acquired toxicity. Although many studies have indicated that mitochondria are deeply involved in mutant SOD1-mediated toxicity, the mechanisms through which mutant SOD1 causes neurodegenration are uncertain. Recently, it has been revealed that the mitochondria-associated membrane (MAM), which is a specialized subdomain of ER very close to mitochondria, is essential for mitochondrial functions and cellular homeostasis. To examine the possible involvement of MAM dysfunction in mutant SOD1-mediated neurodegeneration, we fractionated MAM from spinal cord extracts of SOD1G93A transgenic mice and cultured neuroblastoma Neuro2a cells. We found that the mutant SOD1 was abnormally accumulated at MAM. Interestingly, the amount of accumulated mutant SOD1 at MAM rapidly increased on the presymptomatic stage, but significantly decreased at the disease onset. While, the level of mutant SOD1 continuously increased in the other organelle fractions, such as mitochondria. We also found that mutant SOD1 did not accumulate at MAM in livers and primary cultured astrocytes of SOD1G93A transgenic mice, suggesting that accumulation of mutant SOD1 at MAM is observed specifically in neurons. Moreover, over-expression of fusion-in-sarcoma (FUS) or TAR DNA-binding protein-43 (TDP-43) mutants, genes causative for ALS, induced an abnormal accumulation of wild-type SOD1 at MAM in Neuro2a cells, implying the misfolded SOD1 accumulation at MAM is also involved in non-SOD1 ALS. These data suggest that dysfunction of MAM caused by the mutant or misfolded SOD1 accumulation might be tightly correlated with neurodegeneration in ALS.	P1-1-216 脊髄小脳変性症3型マウスモデルにおけるmGluR1の機能不全とAAV9を用いた遺伝子治療によるその機能回復 Deficient metabotropic glutamate receptor signaling and rescue through intravascular administration of AAV9 in a mouse model of spinocerebellar ataxia type 3 ○今野歩1, , 三宅紀子2, 三宅弘一2, 柳茂3, 島田隆2, 平井宏和1 ○Ayumu Konno1, Anton N Shuvaev1, Noriko Miyake2, Koichi Miyake2, Shigeru Yanagi3, Takashi Shimada2, Hirokazu Hirai1 群馬大院・医・神経生理1, 日本医科大・医生化・分子生物（分子遺伝）2, 東京薬科大・生命科学部・分子生化学3 Dept Neurophysiol, Gunma Univ, Gunma1, Dept Biochem & Mol Biol, Nippon Med Sch, Tokyo2, Lab of Mol Biochem, Sch of Life Sci, Tokyo Univ of Pharma and Life Sci, Tokyo3 Spinocerebellar ataxia type 3 (SCA3) is caused by the abnormal expansion of CAG repeats within the ataxin-3 gene. Previously, we generated transgenic mice (SCA3 mice) that express a truncated form of ataxin-3 containing abnormally expanded CAG repeats specifically in cerebellar Purkinje cells (PCs). These SCA3 mice show severe ataxia and atrophy of the cerebellum. Here, we further characterized these SCA3 mice. Analysis of PCs from advanced stage SCA3 mice revealed a significant decrease in membrane capacitance (Cm) due to poor dendritic arborization and the loss of synaptically evoked suppression of excitation (SSE), which is triggered by metabotropic glutamate receptor subtype1 (mGluR1)-mediated endocannabinoid production, with an overall preservation of AMPA receptor-mediated fast synaptic transmission. These cerebellar phenotypes are reminiscent of retinoic acid receptor-related orphan receptor α (RORα)-defective staggerer mice, and consistent with this, the PCs of the SCA3 mouse also show a marked decrease in RORα. This suggests that a reduction in RORα underlies the cerebellar phenotypes of SCA3 mice. To rescue SCA3 mice, we intravenously injected adeno-associated viral vector serotype-9 (AAV9) expressing CRAG, a molecule that facilitates the degradation of stress proteins, which transduced PCs primarily in the lobule X. Concomitant with the development of their dendritic tree, transduced PCs showed a significantly larger Cm compared to non-transduced PCs and showed the expression of SSE following mGluR1 activation. These results suggest that mutant ataxin-3 causes defects in dendritic differentiation and mGluR-signaling in SCA3 mouse PCs, which could be, at least partially, prevented by neonatal intravenous injections of AAV9 expressing CRAG.
P1-1-217 神経変性疾患病態における複製依存的DNA修復の関与 Replication-dependent DNA repair in SCA1 pathology ○田村拓也1, , 藤田啓太1, 伊藤日加瑠1, 本木和美1, 島村徹平3, 勝田明寿香4, 塩飽裕紀1, 曽根雅紀4, 田川一彦1, 井元清哉3, 宮野悟3, 岡澤均1 ○Takuya Tamura1, Sam S Barclay2, Kyota Fujtia1, Hikaru Ito1, Kazumi Motoki1, Teppei Shimamura3, Asuka Katsuta4, Hiroki Shiwaku1, Masaki Sone4, Kazuhiko Tagawa1, Seiya Imoto3, Satoru Miyano33, Hitoshi Okazawa1 東京医科歯科大学1, 東京大医科研3, 東邦大学4 Dept of neuropathology, Tokyo Medical and Dental University, Tokyo1, Imperial college, London, UK2, Institute of Medical Science, The University of Tokyo, Tokyo, Japan3, Toho Univ, Chiba, Japan4 Recent research has suggested DNA repair genes to be critical regulators of genotoxic stress signals in polyglutamine (polyQ) disease pathogenesis. Our previous results indicated that attenuation of Ku70 dependent non-homologous end joining (NHEJ) is critically involved in the pathology of Huntington's disease (HD). NHEJ is the main DNA repair pathway in non-dividing neuronal cells because it occurs in a DNA replication independent manner. Here, we found expression of mutant ataxin-1 (Atxn1) similarly induced DNA double strand break (DSB) in Drosophila. In addition, mutant Atxn1's effect in mouse cerebellar neurons on levels of DSB DNA damage marker γH2AX was confirmed. However, Ku70 did not sufficiently recover the phenotype as seen in HD fly models. Therefore, a systematic in vivo screen of all available Drosophila melanogaster homolog DNA repair genes was performed based on the effect of their over-expression on lifespan as well as developmental viability assessment in human Spinocerebellar Ataxia Type 1 (SCA1) Drosophila models expressing mutant Atxn1. Surprisingly, the result of the screening suggested that recombination-dependent DNA repair (which should not occur at non-dividing neuron) is related to SCA1 pathology. Our further analysis is elucidating this discrepancy.	P1-1-218 CalpainがALSのTDP-43病理形成に重要な役割を果たす Calpain plays a crucial role in TDP-43 pathology in ALS ○山下雄也1,2,3, 日出山拓人3, 寺本さやか1,2,3, 高野二郎4, 岩田修永4,5, 西道隆臣4, 郭伸1,2,3,6 ○Takenari Yamashita1,2,3, Takuto Hideyama3, Sayaka Teramoto1,2,3, Jiro Takano4, Nobuhisa Iwata4,5, Takaomi C Saido4, Shin Kwak1,2,3,6 東京大学大学院　医学系研究科　疾患生命工学センター　臨床医工学部門1, 東京大学大学院　医学系研究科 脳神経医学専攻　神経内科学3, 理化学研究所　脳科学総合研究センター4, 長崎大学　医歯薬学総合研究科5, 国際医療福祉大学　臨床医学研究センター6 Div Clin Biotechnol, Cent Dis Biol Integr Med Grad Schl Med, Univ of Tokyo, Tokyo1, CREST, JST2, Dept Neurol, Med Grad Schl Med, Univ of Tokyo, Tokyo3, Lab Proteolyt Neurosci, RIKEN BSI4, Lab Mol Biol Biotech, Dept Mol Med Sci, Grad Schl Biomed Sci, Nagasaki Univ5, Clin Res Cent Med, Intnatl Univ Health Welfare6 Motor neurons of sporadic amyotrophic lateral sclerosis (ALS) patients exhibit altered processing of TAR DNA-binding protein (TDP-43) and inefficient RNA editing at the GluA2 glutamine/arginine (Q/R) site, both of which are the disease-specific molecular abnormalities. RNA editing at GluA2 Q/R site is specifically catalyzed by adenosine deaminase acting on RNA 2 (ADAR2), and motor neurons devoid of ADAR2 activity express abnormally Ca2+-permeable AMPA receptors that contain Q/R site-unedited GluA2 and undergo neuronal death in conditional ADAR2 knockout (AR2) mice, in which the ADAR2 gene is targeted in cholinergic neurons including spinal motor neurons. Moreover, both ADAR2 underactivity and TDP-43 pathology are concomitantly observed in the same motor neurons of sporadic ALS patients, suggesting a molecular link between these two death-causing molecular abnormalities, but how they link each other is poorly understood.We found that TDP-43 was cleaved into fragments and disappeared from the nucleus with formation of aggregates in the cytoplasm of the motor neurons devoid of ADAR2 in heterozygous AR2 mice. Calpain, a Ca2+-dependent serine protease, was activated in the AR2 mouse spinal cord and calpain specifically cleaved TDP-43 into aggregation-prone fragments in the lysates of cultured cells. Moreover, the TDP-43 mislocalization was inhibited when the Ca2+ influx through the AMPA receptors was normalized in AR2 mice, or when calpastatin, the endogenous calpain inhibitor, was overexpressed. On the contrary, TDP-43 mislocalization was exaggerated when calpastatin was knocked out in the mice. Calpain cleaved TDP-43 at several positions in the C-terminal region, and calpain-dependent TDP-43 fragments were demonstrated in the brains and spinal cords of ALS patients. Taken together, the calpain-dependent cleavage of TDP-43 plays a crucial role in ALS pathology.
P1-1-219 組換えウイルスベクターによるラット・マウス運動ニューロンへの遺伝子導入 Adenovirus-, lentivirus-, and adeno-associated virus-mediated gene transfer to adult rodent motoneurons in vivo ○渡部和彦1, 秋山けい子1, 河上江美子1, 石井智裕1, 柳澤比呂子1, 三五一憲1, 塚本雅美1, 岡戸晴生2, 三輪昭子2, 三宅弘一3 ○Kazuhiko Watabe1, Keiko Akiyama1, Emiko Kawakami1, Tomohiro Ishii1, Hiroko Yanagisawa1, Kazunori Sango1, Masami Tsukamoto1, Haruo Okado2, Akiko Miwa2, Koichi Miyake3 東京都医学総合研究所　神経変性病理1, 東京都医学総合研究所　神経細胞分化2, 日本医科大学　生化学・分子生物学3 ALS and Neuropathy Pro., Tokyo Metropol. Inst. Med. Sci.1, Neuronal Development Pro., Tokyo Metropol. Inst. Med. Sci.2, Dept. Biochem. Mol. Biol., Nippon Med. Sch.3 We have previously demonstrated that combined adenoviral expression of TDP-43 and FUS genes with shRNAs for proteolytic pathways enhances cytoplasmic aggregate formation in adult rat facial motoneurons in vivo. In this study we produced recombinant adenovirus and lentivirus vectors encoding wild type and mutant TDP-43 or FUS, and those encoding shRNAs for proteasome (PSMC1), endosome (VPS24, TSG101) and autophagy (ATG5) systems, and adeno-associated virus type 2, 6, and 9 (AAV2, 6, and 9) expressing GFP, to examine their transduction ability of the foreign genes in adult rat and mouse facial motoneurons. Transduction efficiency of adenoviruses against facial motoneurons via retrograde transport by facial nerve injections was much higher in mouse than in rat, and cytoplasmic aggregate formation was readily demonstrated in both species by co-infection of adenovirus encoding shRNA for the proteolytic pathways with TDP-43 or FUS adenovirus. Stereotactic injection of recombinant lentiviruses into the rat brain stem facial nuclei induced high expression of the foreign genes in both facial motoneurons and surrounding glial cells. When AAV2, 6, or 9-GFP was injected into rat or mouse facial nerve, strong GFP expression in facial motoneurons was achieved in mice with AAV6 or 9-GFP treatment. We are currently examining the aggregate formation of TDP-43 and FUS using lentivirus and AAV in mouse facial motoneurons.	P1-1-220 ハンチントン病モデルマウスにおけるCAGリピートの不安定性とhip1r遺伝子の発現様式の関連性について Correlation between CAG repeat instability in Huntington's disease model mice and the expression pattern of the hip1-rerated gene in the murine brain ○増田知之1, 佐久間千恵2, 上野孝之3, 山岸敏之4, 大桃秀樹1, 八木沼洋行2, 上田秀一1 ○Tomoyuki Masuda1, Chie Sakuma2, Takayuki Ueno3, Toshiyuki Yamagishi4, Hideki Ohmomo1, Hiroyuki Yaginuma2, Shuichi Ueda1 獨協医大・医・ 解剖学（組織）1, 福島県立医大・医・神経解剖・発生学2, 仙台医療センター3, 大坂市立大院・医学研究科4 Dept Histol Neurobiol, Dokkyo Med Univ, Tochigi1, Dept Anat, Fukushima Med Univ, Fukushima2, Sendai Med Center, Miyagi3, Dept Anat, Grad Sch Med, Osaka City Univ, Osaka4 Huntington's disease (HD) is a dominant neurodegenerative disorder caused by the expansion of CAG repeats in the gene encoding huntingtin. A previous study revealed that both elderly HD model mice and HD patients has somatic CAG repeat instability in the specific region of the brain. Here we report the correlation between the mRNA expression pattern of the huntingtin interacting protein 1-related (hip1r) gene and the region of CAG repeat instability in the mouse head. We closely examined the hip1r expression by in situ hybridization in the murine head from embryonic day 13 to postnatal day 0. Hip1r was expressed in secretory glands such as a submandibular gland and nasal glands. Furthermore, hip1r was highly expressed in the hippocampus, pons, olfactory system and cortex where CAG repeat instability occurred. In contrast, hip1r expression was relatively weak in the cerebellum where the shorter CAG repeat length was observed in HD model mice and HD patients. These findings suggest the hypothesis that the hip1r expression in the young murine brain may be involved in instability of CAG repeats in HD model mice and HD patients after aging.
P1-1-221 熱ショック因子1は球脊髄性筋萎縮症の運動神経変性を抑える Heat shock factor-1 (Hsf-1) suppresses motor neuron degeneration in spinal and bulbar muscular atrophy (SBMA) ○近藤直英1, 勝野雅央1, 足立弘明1, 南山誠1, 土井英樹1, 松本慎二郎1, 宮崎雄1, 飯田円1, 中辻秀朗1, 藤内玄規1, 石垣診祐1, 藤岡祐介1, 渡辺宏久1, 田中章景1, 祖父江元1 ○Naohide Kondo1, Masahisa Katsuno1, Hiroaki Adachi1, Makoto Minamiyama1, Hideki Doi1, Shinjiro Matsumoto1, Yu Miyazaki1, Madoka Iida1, Hideaki Nakatsuji1, Genki Tohnai1, Shinsuke Ishigaki1, Yusuke Fujioka11, Hirohisa Watanabe1, Fumiaki Tanaka1, Gen Sobue1 名古屋大学大学院　医学系研究科　神経内科学1 Dept Neurol, Univ of Nagoya, Nagoya1 Objective: Late-onset neurodegenerative disorders have an essential feature that abnormal proteins accumulate in specific regions, whereas the disease-causing mutant genes are broadly expressed. Heat shock factor-1 (Hsf-1) induces the expression of Hsps, such as Hsp70, molecular chaperones that play a protective role in the neurodegenerative process by refolding and solubilizing pathogenic proteins. To elucidate the role of Hsf-1 in pathological lesion selectivity of spinal and bulbar muscular atrophy (SBMA), a adult-onset motor neuron disease, here we investigate the effect of Hsf-1 on the pathogenesis of the SBMA mouse model. Methods: We performed immunohistochemistry and western blotting of various tissues from wild-type; AR-97Q (SBMA model mouse: 97Q Tg/-, Hsf-1 +/+); and AR-97Q Hsf-1 +/- (Hsf-1 knockout SBMA mouse: 97Q Tg/-, Hsf-1 +/-) mice using anti-Hsf-1, anti-Hsp70 and 1C2 antibodies. Moreover, we analyzed the effect of lentiviral over-expression of HSF-1 in this mouse model. Results: In Hsf-1 knockout SBMA mice, abnormal androgen receptor (AR) accumulates in the cerebral visual cortex, liver and pituitary, which are not affected in their genetically unmodified counterparts. In the spinal anterior horn and other part of central nervous system of AR-97Q Hsf-1 +/- mice, the accumulation of mutant AR increased significantly through Hsp70 down-regulation. Furthermore, in the motor cortex and striatum, the frequency of pathogenic AR accumulation around the lentiviral vector-injected area where HSF-1 was highly expressed was decreased in comparison with that in the contralateral side without treatment. In addition, the neuron sizes of the motor cortex and striatum were significantly increased by the HSF-1 injection. Conclusions: These results suggest that Hsf-1 contributes to the determination of the pathological lesion selectivity in SBMA.	P1-1-222 筋萎縮性側索硬化症関連タンパク：TDP-43封入体の形成―マウス胎仔電気穿孔法による大脳皮質運動野への疾患関連遺伝子の導入 ALS associated pathogenic protein: TDP-43 aggregation in mice motor area of cerebral cortex by in utero electroporation ○赤松恵1, 詫間浩1, 山下雄也2, 岡田拓也3, 石井一弘1, 桝和子3, 郭伸2, 桝正幸3, 玉岡晃1 ○Megumi Akamatsu1, Hiroshi Takuma1, Takenari Yamashita2, Takuya Okada3, Kazuhiro Ishii1, Kazuko Keino-Masu3, Shin Kwak2, Masayuki Masu3, Akira Tamaoka1 筑波大院・医学医療・神経内科1, 東京大院・疾患生命工学・臨床医工学2, 筑波大院・医学医療・分子神経生物3 Dept Neurol, Univ of Tsukuba, Ibaraki1, Div of Clinic Biotechnol, Disease Biol and Integrative Med, Univ of Tokyo, Tokyo2, Dept of Molecular Neurobiol, Univ of Tsukuba, Ibaraki3 TDP-43 is a discriminative protein that is found as intracellular aggregations in neurons of cerebral cortex and spinal cord of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) patients. In aggregations, TDP-43 exists as phosphorylated, C-terminally cleaved and ubiquitinated. The upper and lower motor neuronal loss is a major pathology of ALS, however, the mechanisms of neuronal loss and the relation to the aggregations are still unclear. In this study, we generated a useful model to produce TDP-43 aggregations in the motor cortex using in utero electroporation to mouse embryos, and to clarify the mechanisms of generation of TDP-43 aggregations. Timed pregnant mice on postcoitum day 12.5 were used for electroporation. The plasmids used were full-length TDP-43 and C-terminally fragmented TDP-43 (wild-type or M337V mutant), and all kinds of TDP-43 was tagged GFP. The electroporated mice brains at embryonic day 15.5 and post natal days 7 and 21 were performed immunohistochemical analysis and western blot analysis. In the result, in the case of full-length TDP-43, regardless of whether wild-type or mutant, electroporated TDP-43 localized mostly in the nucleus and aggregations were not observed. In contrast, TDP-43 aggregations were generated in the brains which were electroporated with wild-type or mutant form of C-terminally fragmented TDP-43. These aggregations distributed diffusely not only in the nucleus but also in the cytoplasm and these inclusion bodies were ubiquitinated, and included phosphorylated TDP-43, which represented human pathology of ALS. These results suggest that C-terminally fragmented TDP-43 would be enough to make inclusion bodies. This model using in utero electroporation of pathogenic genes into mouse brain, will become a useful model for studying ALS and may be applicable to other neurodegenerative diseases.

上部に戻る

前に戻る