ポスター発表 (Poster Sessions)

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
P2-1-197 変異SOD1によるラット神経変性モデル脊髄における微小血管壁細胞 Microvascular pericytes of the spinal cord with mutant SOD1-induced neurodegeneration in rats ○割田仁¹, 水野秀紀¹, 加藤昌昭¹, 鈴木直輝¹, 青木正志¹ ○Hitoshi Warita¹, Hideki Mizuno¹, Masaaki Kato¹, Naoki Suzuki¹, Masashi Aoki¹ 東北大学大学院医学系研究科神経内科学¹ Dept Neurol, Univ of Tohoku, Sendai, JAPAN¹ Amyotrophic lateral sclerosis (ALS) is a devastating disease characterized by adult-onset motor neuron degeneration without effective treatment to date. Approximately 2% of ALS cases are linked to mutations in the SOD1 gene. Recent reports have shown disruption of blood-spinal cord barrier (BSCB) in transgenic rodent models and autopsy cases of ALS. Pericytes are known to play a pivotal role in maintaining BSCB. Here, we focused on microvascular pericytes in the spinal cord of a transgenic rat with ALS-linked mutant SOD1 gene. In order to clarify a possible role of the pericytes under the neurodegenerative condition, we examined microvascular morphology and BSCB components such as pericytes, endothelial cells, astrocytes and barrier antigens in the spinal cord of His46Arg mutant SOD1 transgenic (Tg) rats at pre-, early, and late symptomatic stages with their age-matched non-transgenic (non-Tg) littermates. Based on the results, a growth factor for pericytes or vehicle was continuously infused into subarachnoid space of early symptomatic animals for 14 days, followed by multiple immunohistochemistry employing cell-selective markers for the BSCB components in lumbar spinal cord cryosections. In addition, we compared neuropathology in the spinal ventral horns between treated and non-treated Tg rats. In contrast to non-Tg rats, the Tg rats showed a significant and progressive reduction of pericyte-attached microvasculature at the site of neurodegeneration in the ventral spinal cord. In the spinal ventral horn of treated Tg rats, multiple immunohistochemistry revealed significant increase in angiogenesis, BSCB protection, and neuroprotective effects against the ALS-like motor neuron degeneration. Our data suggest that BSCB-protective reagents could rescue the spinal motor neurons in mutant SOD1-induced neurodegeneration. Moreover, microvascular pericytes may be considered as a potential therapeutic target for ALS.	P2-1-198 Optineurin のノックダウンによってNF-κBを介した神経細胞死を認めた Optineurin suppression causes neuronal cell death via NF-κB pathway ○秋月真由美¹, 山下博史¹, 植村健吾¹, 丸山博文², 川上秀史², 伊東秀文¹, 高橋良輔¹ ○Mayumi Akizuki¹, Hirofumi Yamashita¹, Kengo Uemura¹, Hirofumi Maruyama², Hideshi Kawakami², Hidefumi Ito¹, Ryosuke Takahashi¹ 京都大学大学院　医学研究科　臨床神経学¹, 広島大学　原爆放射線医科学研究所² Department of Neurology, Kyoto University Graduate School of Medicine¹, Department of Epidemiology, Research Institute for Radiation Biology and Medicine² Amyotrophic lateral sclerosis (ALS) is an adult-onset neurodegenerative disorder characterized by the progressive degeneration of motor neurons in the brain and spinal cord. The cause of sporadic ALS is unknown, and there is no effective therapy. Mutations in more than 10 genes are reported to cause familial ALS. Among these genes, optineurin (OPTN) is virtually the only gene that is considered to cause classical ALS by a loss-of-function mutation. It is known that wild-type optineurin suppresses NF-κB-activity, but ALS-causing mutant OPTN is unable to suppress NF-κB-activity. It indicates that inappropriate NF-κB activation is the pathogenic mechanism underlying OPTN mutation-related ALS.Therefore, we knocked down OPTN in neuronal cells and examined the resulting NF-κB activity and phenotype. First, we found that NF-κB activity was increased in OPTN-knockdown cells, which was consistent with the previous report that ALS-causing OPTN mutations failed to suppress NF-κB activity. Then we found that OPTN knockdown caused neuronal cell death, and it was inhibited by the pharmacological treatment for NF-κB suppression. We also investigated the downstream molecule of NF-κB. Our study showed that NF-κB is the key molecule in OPTN-mediated ALS.
P2-1-199 家族性ALSモデルの神経炎症におけるトランスグルタミナーゼ2介在性SOD1オリゴマーの関与 The involvement of transglutaminase2 mediated mutant SOD1 oligomer in neuroinflammation of familial ALS model ○大野美樹^1,2, 松本紋子³, 谷口直之³, 高橋良輔¹, 漆谷真² ○Miki Oono^1,2, Ayako Matsumoto³, Naoyuki Taniguchi³, Ryosuke Takahashi¹, Makoto Urushitani² 京都大学大学院　医学系研究科　臨床神経学¹, 滋賀医科大学分子神経科学研究センター神経難病治療学分野², 理化学研究所基幹研究所システム糖鎖生物学研究グループ³ Dep.Neurology, Kyoto Univ, Kyoto, Japan¹, Unit for Neurobiology and Therapeutics, Molecular Neuroscience Research Center (MNRC), Shiga Univ. of Medical Science, Otsu, Japan.², System Glycobiology Research Group, RIKEN, Wako, Japan³ Introduction: We previously reported that transglutaminase 2(TGM2), an endogenous cross-linker, induced oligomer formation of misfolded SOD1 proteins in vitro. It was also shown that TGM2 was upregulated in the spinal cord of mutant SOD1 Tg mice. In this study, we first investigated the interaction between mutant SOD1 and TGM2 in transfected cells. Next, we tested the therapeutic benefit of intrathecal inhibition of TGM2 in the ALS model mice, aiming to target SOD1 oligomers. Method: 1) We co-transfected HEK293A cells with FLAG-tagged SOD1(WT, mutant) and HA-tagged TGM2. Lysates of the transfected cells were immunoprecipitated with anti-FLAG or anti-HA antibody, and were analyzed by Western blotting using anti-HA or anit-SOD1 antibody, respectively. 2) We performed intrathecal introduction of cystamine, an inhibitor of TGM2, using osmotic minipump, which provided a constant amount of cystamine for 6 weeks. Spinal cords were analyzed by Western blotting to quantify SOD1 oligomers and inflammatory factors. Results: 1) Immunoprecipitation assay showed that TGM2 interacted with mutant SOD1 proteins, but not with the WT. 2) The cystamine treatment significantly reduced the amount of oligomeric SOD1 and inflammatory factors such as Mac2, a marker for activated microglia. These data indicate that TGM2 discriminates ALS-relevant misfolded forms of SOD1 from native species in cells, and causes neuroinflammation in ALS model mice through the effect of oligomerization of SOD1.	P2-1-200 運動神経変性疾患におけるスプライソソーム異常 Spliceosome integrity is a common target for motor neuron disease ○築地仁美¹, 井口洋平², 古屋亜佐子¹, 片岡礼音¹, 初田裕幸³, 熱田直樹², 田中章景², 橋詰良夫⁴, 赤津博康⁴, 村山繁雄³, 祖父江元², 山中宏二¹ ○Hitomi Tsuiji¹, Youhei Iguchi², Asako Furuya¹, Ayane Kataoka¹, Hiroyuki Hatsuta³, Naoki Atsuta², Fumiaki Tanaka², Yoshio Hashizume⁴, Hiroyasu Akatsu⁴, Shigeo Murayama³, Gen Sobue², Koji Yamanaka¹¹ 理化学研究所・脳センター・運動ニューロン変性¹, 名古屋大学・医・神経内科², 東京都健康長寿医療センター³, 福祉村病院　長寿医学研究所⁴ Lab for Motor Neuron Disease, RIKEN BSI, Saitama¹, Dept of Neurol., Nagoya Univ., Nagoya², Dept of Neuropathol., Tokyo Metro Inst of Geront., Tokyo³, Choju Medical Inst., Fukushimura Hospital, Aichi⁴ Neurodegenerative diseases are characterized by the death of specific type of neurons. Two motor neuron diseases, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are caused by distinct genes involved in RNA metabolism, TDP-43 and FUS/TLS, and SMN, respectively. However, whether there is a shared defective mechanism in RNA metabolism common to these two diseases remains unclear. We show that TDP-43 and FUS/TLS localize in nuclear gems through an association with the SMN, and that all three genes function in spliceosome maintenance. We show that in ALS, gems are lost, U snRNA levels are up-regulated and spliceosomal U snRNPs abnormally and extensively accumulate in motor neuron nuclei, but not in the temporal lobe of FTLD with TDP-43 pathology. These findings indicate that a profound loss of spliceosome integrity is a critical mechanism common to neurodegeneration in ALS and SMA, and may explain cell-type specific vulnerability of motor neurons.
P2-1-201 脱ユビキチン化酵素USP15によるTDP-43ならびにSMNの制御 Loss of USP15 alters TDP-43 and SMN properties associated with neurodegenerative disorders ○鶴田文憲¹, , 千葉智樹¹ ○Fuminori Tsuruta¹, Jaehyun Kim¹, Tomoki Chiba¹ 筑波大学　生命環境系¹ Grad Sch of Life and Env Sci, Univ of Tsukuba, Japan¹ Ubiquitin specific protease, USP15, is involved in a variety of cellular functions through deubiquitination of target proteins. Recent studies have reported that expression level of USP15 is decreased in spinocerebellar ataxia mice, implying that USP15 is associated with regulation of neuronal functions. However, the precise mechanisms of how USP15 regulates nervous system remains elusive. In this study, we report that USP15 is a novel mediator that links dysfunction of RNA binding proteins to neurodegenerative disorders. We found that USP15-deficient mice exhibit limb-clasping reflexes and abnormal morphology of purkinje cells in an age-dependent manner. In addition, proteomics analysis revealed that USP15 interacts with several RNA binding proteins. Furthermore, loss of USP15 changes TDP-43 and SMN properties, which are regulate RNA turnover and responsible for several neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), frontotemporal lobar degeneration (FTLD) and spinal muscular strophy (SMA). Taken together, our data suggest that USP15 plays crucial roles in the regulation of TDP-43 and SMN, and provide a novel mechanism by which RNA binding proteins are associated with neurodegenerative disorders.	P2-1-202 脊髄小脳変性症6型モデルマウス小脳の網羅的遺伝子発現解析 Gene expression profiles in the cerebellum of SCA6 mouse models ○相川知徳^1,2, 茂櫛薫³, 飯島久美子³, 田中博³, 水澤英洋^1,2,4, 渡瀬啓^1,2 ○Tomonori Aikawa^1,2, Kaoru Mogushi³, Kumiko Iijima³, Hiroshi Tanaka³, Hidehiro Mizusawa^1,2,4, Kei Watase^1,2 東京医科歯科大学脳統合機能研究センター¹, CREST、科学技術振興機構², 東京医科歯科大学情報医科学センター³, 東京医科歯科大学脳神経病態学⁴ The Center for Brain Integration Research, Tokyo Medical and Dental Univ, Tokyo¹, CREST, JST, Japan², Information Center for Medical Sciences, Tokyo Medical and Dental Univ, Tokyo³, Dept Neurology and Neurogical Science, Tokyo Medical and Dental Univ, Tokyo⁴ Spinocerebellar ataxia type 6 (SCA6) is one of nine known dominantly inherited neurodegenerative disorder caused by the expansion of a CAG repeat. In SCA6, the CAG repeat encodes a polyQ tract in the Ca_V2.1 subunit. Molecular pathogenesis of SCA6 remains to be elucidated. Although Ca_V2.1 is expressed widely in the brain, the mutation causes selective degeneration of cerebellar Purkinje cells (PCs) and inferior olivary neurons, possibly through a toxic gain-of function mechanism.We hypothesized that examination of molecular changes occurring in the cerebellum of SCA6 animal models at their early symptomatic stage could reveal the molecular pathways that could be targeted to modulate or monitor the pathogenesis. To this end, here we surveyed cerebellar gene expression patterns by microarray analysis in two faithful Sca6 knockin models, Sca6-MPI-118Q and Sca6^84Q, with the former having longer repeat size and showing earlier onset of ataxia and more distinct PC degeneration compared to the latter (Unno et al., PNAS 2012). In the cerebellum of 6-week-old homozygous MPI-118Q mice, we found that 376 genes were upregulated while 394 genes were downregulated in compared with control. qPCR analysis confirmed most of these changes. Among these alterations, 167 genes were replicated in the cerebellum of 20 months old Sca6^84Q/84Q mice. Interestingly, these shared changes include upregulation of several genes involved in oxidative stress and neuroinflammation.We also compared the data with those obtained from Sca1^154Q/2Q and Sca7^266Q/7Q (Gatchel et al., PNAS 2008) to discover the common molecular changes shared among different types of SCAs. We found that only three probe sets were commonly misregulated among four models.Taken together, these results suggest different types of SCAs may share a few common early transcriptional alterations in the cerebellum and neuroinflammatory response and enhanced oxidative stress may be unique early events occurring in SCA6 cerebellum.
P2-1-203 TDP-43の核外脱出シグナル内に存在する酸性アミノ酸がその構造と機能維持に及ぼす役割について Conserved acidic amino acid residues in the nuclear export signal of the RNA interacting domain regulates conformation and function of TDP-43 ○漆谷真¹, 小代明美¹, 綾木孝², 藤原範子³, 伊東秀文⁴ ○Makoto Urushitani¹, Akemi Shodai¹, Takashi Ayaki², Noriko Fujiwara³, Hidefumi Ito⁴ 滋賀医科大学・分子神経科学研究センター・神経難病治療学¹, 兵庫医科大学生化学², 京都大学大学院医学研究科　神経内科³, 和歌山県立医科大学　神経内科⁴ Mol Neurosci Res Centr, Shiga Univ of Med Sci, Otsu¹, Dept of Biochem, Hyogo Med College, Nishinomiya², Dept of Neurol, Kyoto Univ Sch of Med, Kyoto³, Dept Neurol, Wakayama Med Univ Grad Sch of Med, Wakayama⁴ The current understanding of ALS pathogenesis is based on results suggesting RNA mishandling and protein misfolding of TAR DNA-binding protein 43 kDa (TDP-43). However, the molecular links between RNA interacting domains (RRMs) and ALS pathologies remain elusive. We investigated the roles of two acidic amino acid residues, Glu246 (E246) and Asp247 (D247), in the nuclear export signal of RRM2 in the conformation and function of TDP-43, based on the previous crystal study, describing the intermolecular association upon DNA interaction. First, we unexpectedly found that the RRM2 domain in phosphate-buffered saline is a stable monomer regardless of DNA interaction by gel filtration analysis. Moreover, studies using substitution mutants at Glu246 and Asp247 displayed that these residues, especially Asp247 plays a crucial role in the preservation of the functional RRM2 monomers. In particular, substitution to glycine at Asp247 and/or Glu246 promoted the formation of fibrillar oligomers of RRM2 accompanied by the loss of DNA binding affinity, which also affected the conformation and the RNA splicing function of full-length TDP-43. Furthermore, a novel monoclonal antibody targeting residues containing Asp247 stained with TDP-43 inclusions of ALS patients and mislocalized cytosolic TDP-43 in cultured cells, but not nuclear wild-type TDP-43. Our findings indicate that Glu246 and Asp247 play a pivotal role in the proper conformation and function of TDP-43. Especially, Asp247 should be focused as molecular target showing aberrant conformation related to TDP-43 proteinopathy.	P2-1-204 細胞特異的トランスクリプトームを用いた、弧発性ALS患者脊髄のDNAマイクロアレイによる解析 Microarray analysis in spinal cords of sporadic ALS patients with cell-type specific transcriptome ○山下博史^1,2, 藤森典子², 伊東秀文¹, 井口洋平³, 熱田直樹³, 田中章景³, 祖父江元³, 高橋良輔¹, 山中宏二² ○Hirofumi Yamashita^1,2, Noriko Fujimori², Hidefumi Ito¹, Yohei Iguchi³, Naoki Atsuta³, Fumiaki Tanaka³, Gen Sobue³, Ryosuke Takahashi¹, Koji Yamanaka² 京都大学大学院　医学系研究科　臨床神経学¹, 独立行政法人理化学研究所　脳科学総合研究センター　運動ニューロン変性研究チーム², 名古屋大学大学院　医学系研究科　臨床神経学³ Dept Neurology, Univ of Kyoto, Kyoto¹, RIKEN, BSI, Laboratory for motor neuron disease, Wako, Japan², Dept Neurology, Univ of Nagoya, Nagoya³ Objective: With DNA microarray, we analyze the molecular pathomechanism in sporadic ALS spinal cords with a focus on the function of microglia and astrocytes. We analyzed the microarray data in the cell type specific manner to understand the molecular mechanisms within each cell type of ALS spinal cord.Background: Glial cells including astrocytes and microglia are reported to be actively involved in motor neuron death in ALS, but the precise mechanisms for the "non-cell autonomous neuron death" have not been elucidated.Design/Methods: We profiled using DNA microarray the mRNA expression with cervical spinal cords of 4 sporadic ALS patients and 5 disease-control. To predict the cell type(s) in which each gene was expressed abundantly, we established the cell-type specific transcriptomes using mouse CNS primary culture, then the integrated database was converted to human orthologue. Isolated misregulated genes from microarray was analyzed in terms of glial functions, by using cell-type specific mouse transcriptomes.Results: We isolated 197 genes which were significantly changed in the spinal cords of ALS patients. We then classified these genes according to the cells that expressed those genes abundantly, and found nearly half of those genes were expressed abundantly in microglia or astrocytes. Furthermore, many of these genes were also changed in ALS mouse models (SOD1G37R Tg mice, SOD1G85R Tg mice). We confirmed that the predicted gene expression pattern was true by immunohistochemistry for several genes with spinal cords of ALS mouse models. Pathway analysis predicted that innate immunity was one of the significantly altered pathways in glial cells. Conclusion: We could predict the molecular pathomechamism, especially of glia contributing to the non-cell autonomous motor neuronal death of the ALS in the spinal cord that consists of heterogenous cell types.
P2-1-205 TDP-43変異を有するiPS細胞を用いた疾患由来運動ニューロンの作製 Generation of motor neurons through patient-specific iPSCs with mutant TDP-43 ○江川斉宏^1,2, 高橋良輔³, 井上治久^1,2 ○Naohiro Egawa^1,2, Ryosuke Takahashi³, Haruhisa Inoue^1,2 京都大学iPS細胞研究所¹, JST CREST², 京都大学大学院医学研究科³ Center for iPS Cell Research and Application, Kyoto University, Kyoto, Japan¹, JST-CREST², Graduate School of Medicine, Kyoto University, Kyoto, Japan³ Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disorder in which motor neuron (MN) loss in the spinal cord leads to progressive paralysis and death. Cytosolic aggregations in ALS MNs are composed of Tar DNA-binding protein-43 (TDP-43). Genetic analysis has identified more than twenty mutations of TDP-43 in ALS cases. Although accumulating evidence provides several hypotheses of disease mechanism, it is still needed to discover effective cure for ALS. We aimed to reveal cellular phenotypes in ALS MNs for identifying a drug-screening target for ALS using patient-specific iPSCs. To generate patient-specific iPSCs, dermal fibroblasts were obtained by biopsy from ALS patients carrying mutant TDP-43. The fibroblasts were reprogrammed by retrovirus or episomal vectors. Disease-specific iPSCs were differentiated into MNs expressing HB9 and SMI-32. Despite short culture period, ALS MNs recapitulated several disease phenotypes observed in patient and animal models. Disease-specific iPSCs might provide a first step for drug-screening platform for ALS using patient-specific iPSCs.	P2-1-206 家族性ALS関連遺伝子FUS/TLSのアルギニン・メチル化の核-細胞質シャトリング機構への関与 The effect of arginine methylation on the nuclear-cytoplasmic shuttling of FUS/TLS ○藤井早紀子¹, 高梨啓介¹, 坂本宗樹¹, 北城敬子¹, 山口淳¹ ○Sakiko Fujii¹, Keisuke Takanashi¹, Muneki Sakamoto¹, Keiko Kitajo¹, Atsushi Yamaguchi¹ 千葉大学大学院医学研究院　神経生物学 C1¹ Dept Neurobiology, Univ of Chiba, Chiba¹ Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). FUS/TLS encodes a nuclear-cytoplasmic shuttling RNA binding protein, which is involved in RNA metabolism. Protein arginine methyltransferase 1 (PRMT1) is a binding partner for FUS/TLS primarily in the nucleus of motor neuron. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. The cytoplasmic localization of FUS/TLS P525L, severe type of FUS/TLS-related familal ALS, was rescued by the inhibition of arginine methylation in SH-SY5Y cell. These findings indicate that PRMT1-mediated arginine methylation could regulate the nuclear-cytoplasmic shuttling of FUS/TLS.
P2-1-207 発育鶏胚を用いたポリグルタミン病モデルの構築 Development of a novel model for polyglutamine pathogenesis using the chicken embryo ○柴田昌宏¹, 中山瞳¹, 安戸方邦¹, 伊藤健二朗¹, 植村修², 岡本仁², 佐藤昇¹ ○Masahiro Shibata¹, Hitomi Nakayama¹, Masakuni Yasudo¹, Kenjiro Ito¹, Osamu Uemura², Hitoshi Okamoto², Noboru Sato¹ 新潟大学大学院医歯学総合研究科肉眼解剖学¹, 理化学研究所・脳科学総合研究センター発生遺伝子制御研究チーム² Div Gross Anatomy and Morphogenesis, Niigata Univ, Niigata¹, Lab for Developmental Gene Regulation, RIKEN Brain Science Institute, Saitama² Polyglutamine (polyQ) diseases are dominantly inherited neurodegenerative disorders caused by the expansion of glutamine-encoding repeat within each identified genes. Accumulating evidences suggest that gain-of-function of polyQ proteins, but not loss-of-function of naive proteins, leads to neurodegeneration. To study polyQ pathogenesis in vivo we took advantage of the chicken embryo system which now allows efficient gene transfer into the developing tissues including the nervous system. In the present study, we confirmed the truncated ataxin-3 gene carrying 77 polyQ repeat (Q77-trAT3) are introduced into the neural tube efficiently 24 hrs after transfection of plasmid constructs by in ovo electroporation. Transfer of Q77-trAT3 into the developing motor neurons using the islet-1 enhancer/promotor resulted in appearance of motor neurons expressing diffuse Q77-trAT3 or nucleic aggregates of Q77-trAT3. Motor neurons expressing aggregates of Q77-trAT3 clearly showed atrophied cell bodies a week after introduction of Q77-trAT3. This atrophy was not prevented by the co-expression of anti-apoptotic Bcl-xl or by the chronic blockade of neuromuscular activity by curare, suggesting that polyQ pathogenesis is distinct from the cellular mechanism of programmed neuronal death. Taken together, these results suggest that misexpression of polyQ protein in the chicken embryo can be an alternative model for investigating cellular pathogenesis of polyQ proteins in vivo.	P2-1-208 球脊髄性筋萎縮症モデルにおけるシャクヤク抽出物の治療効果 A peony extract enhances protein degradation systems and exerts therapeutic effects in the polyglutamine-mediated motor neuron disease ○藤内玄規¹, 足立弘明¹, 勝野雅央¹, 南山誠¹, 土井英樹¹, 松本慎二郎¹, 近藤直英¹, 宮崎雄¹, 田中章景¹, 大塚健三², 祖父江元¹ ○Genki Tohnai¹, Hiroaki Adachi¹, Masahisa Katsuno¹, Makoto Minamiyama¹, Hideki Doi¹, Shinjiro Matsumoto¹, Naohide Kondo¹, Yu Miyazaki¹, Fumiaki Tanaka¹, Kenzo Ohtsuka², Gen Sobue¹ 名古屋大学大学院・医学系研究科・神経内科学¹, 中部大学・応用生物・環境生物² Dept Neurol, Nagoya Univ, Nagoya¹, Dept Environ Biol, Chubu Univ, Kasugai² Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by the expansion of a trinucleotide CAG repeat in the causative genes. One of these is spinal and bulbar muscular atrophy (SBMA), characterized by premature muscular exhaustion, progressive muscular weakness, atrophy, and fasciculation in bulbar and limb muscles. Pathological findings of SBMA are lower motor neuronal loss and diffuse nuclear accumulations and nuclear inclusions (NIs) of polyQ-expanded mutant AR in the residual motor neurons in the brainstem and spinal cord as well as in some other visceral organs. Heat shock protein (Hsp), a stress-induced chaperone, facilitates refolding and degradation of misfolded proteins. We examined the effects of medical-induction of molecular chaperones in the cell and mouse models of SBMA. Peony plants have been used in traditional Chinese medicines or herbal medicines in China and Japan. A peony extract, one of the major constituents of peony plants, upregulated molecular chaperone expression. We administrated the peony extract from age 5 to 30 weeks at doses of 6.7 or 13.4 mg/kg to male AR-97Q or AR-24Q mice and examined various neurological and behavioral parameters. Administration of the peony extract markedly ameliorated motor impairments in SBMA mice without detectable toxicity, and reduced amounts of monomeric and nuclear accumulated mutant AR. Mutant AR was preferentially degraded in the presence of the compound in both SBMA cell and mouse models when compared with wild-type AR. It also significantly induced Hsp70 and Hsp40. These observations suggest that the compound is a promising therapeutic candidate for polyQ-mediated neurodegenerative diseases including SBMA.
P2-1-209 TDP-43プロテイノパチーモデルマーモセットの作出 Establishment of a new marmoset model of TDP-43 proteinopathy ○小林玲央奈¹, 原　宮内央子^1,2, 小澤史子¹, 岡原純子³, 佐々木えりか^1,3, 岡野洋尚ジェイムス^1,2, 岡野栄之¹ ○Reona Kobayashi¹, Chikako Hara-Miyauchi^1,2, Fumiko Ozawa¹, Junko Okahara³, Erika Sasaki^1,3, Hirotaka James Okano^1,2, Hideyuki Okano¹ 慶應大院・医・生理¹, 慈恵医大・医・再生医学², 実中研・応用発生学³ Dept Physiol, Keio Univ, Tokyo¹, Division of Regenerative Medicine, Jikei Univ, Tokyo², Central Institute for Experimental Animals, Kanagawa³ TAR DNA-binding protein 43 kDa (TDP-43) is known as causative gene for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We focused on TDP-43 which has been reported as the sporadic and familial responsible gene of ALS and FTLD. TDP-43 is expressed ubiquitously and normally localized in nuclei. In ALS and FTLD patients, TDP-43 aggregates and generates the inclusions in cytoplasm. In order to further dissect the molecular mechanisms of ALS and FTLD pathology caused by TDP-43 mutations, we decided to utilize model animals. However the flontotemporal lobe is structurally unclear in the rodents, we needed to choose another animal which has higher functional brain. The common marmoset is more similar to humans than rodents in the structure of the brain. Therefore marmoset model of TDP-43 proteinopathy will be able to be a valuable tool for studying the pathogenesis of ALS and FTLD.We decided to generate transgenic marmoset expressing mutated TDP-43. In this presentation, we show the gene constructions of specialized Tg marmoset, expression analysis data of construction in variable cultured cells and in vitro expression and oogenesis data of the construction in marmoset early-stage embryo. In construction, EOS which is early transposon promoter/Oct-4 and Sox2 enhancers, SYN which has activity in neurons, CMV which is cytomegalovirus promoter and CAG which is CMV early enhancer/chicken β actin promoter, were inserted with single or double a vector. In expression analysis, various types of cells, HEK293T, primary neuronal cultured cells and marmoset neurospheres, were studied. And we are checking the oogenesis using early-stage marmoset embryo.	P2-1-210 脊髄小脳失調症2型原因タンパク質Ataxin-2のCdk5によるリン酸化 Phosphorylation of Ataxin-2, a pathological causative gene product of Spinocerebellar ataxia 2, by Cdk5 ○浅田明子¹, 山崎麗奈¹, 三宅真央¹, 斎藤太郎¹, 久永眞市¹ ○Akiko Asada¹, Rena Yamazaki¹, Mao Miyake¹, Taro Saito¹, Shin-ichi Hisanaga¹ 首都大学東京¹ Dept Biol. TMU, Tokyo¹ Spinocerebellar ataxia 2 (SCA2) is a neurodegenerative disorder characterized by progressive ataxia. SCA2 is resulted from poly(Q) expansion in ataxin-2 (Atx2) protein. In SCA2 patients, the poly(Q) repeats are expanded from normal ~22 to more than 32. Atx2 is a ubiquitously expressed cytosolic protein and suggested to have a role in RNA translation and splicing, endocytosis and actin-cytoskeleton organization. Cyclin-dependent kinase 5 (Cdk5) is a Pro-directed Ser/Thr kinase that plays a role in overlap functions of Atx2. Moreover, there are 46 (S/T)P possible Cdk5 phosphorylation sites in Atx2 with 2 (S/T)PX(K/R) best consensus sequence. We hypothesized that Atx2 function is regulated by phosphorylation with Cdk5.Atx2 displayed upward shift of the electrophoretic mobility on SDS-PAGE by coexpression with p25-Cdk5 in COS-7 cells. This mobility shift was diminished with the coexpression of inactive Cdk5, indicated that Atx2 is phosphorylated by Cdk5. To identify the Cdk5 phosphorylation sites, we divided Atx2 into three portions, N-terminal (aa1-507), middle (aa508-905) and C-terminal (aa906-1312) regions, each of which contains 8, 21 or 17 (S/T)P motifs. Cdk5 phosphorylation was observed in the N-terminal and middle fragments with a higher phosphorylation in the middle fragment. Further, these two fragments reduced their protein amounts by coexpression with p25-Cdk5. Since proteasome inhibitor MG132 suppressed the reduction, Cdk5-dependent phosphorylation would be a signal for the proteasomal degradation. To identify the phosphorylation site for degradation, we mutated Thr509 in (S/T)PX(K/R) motif, T509A. Although T509A mutant did not show difference in phosphorylation state, the degradation was suppressed, suggesting that T509 is the phosphorylation site involved in degradation. These are first evidence indicating phosphorylation of Atx2 by Cdk5. Cdk5 phosphorylation could provide the method that controls the protein amounts of poly(Q) mutant SCA2.
P2-1-211 ケラタン硫酸の欠損はALSの早期病態形成を加速させる Ablation of keratan sulfate accelerates early pathogenesis of ALS ○大篭友博¹, 平野健一^1,2, 小林和克^1,2, 松本智宏^1,2, 名取貴光³, 平川晃弘⁴, 今釜史郎², 門松健治¹ ○Tomohiro Ohgomori¹, Kenichi Hirano^1,2, Kazuyoshi Kobayashi^1,2, Tomohiro Matsumoto^1,2, Takamitsu Natori³, Akihiro Hirakawa⁴, Shiro Imagama², Kenji Kadomatsu¹ 名古屋大学大学院　医学系研究科　生物化学講座¹, 名古屋大学医学部附属病院　整形外科², 山梨学院大学健康栄養学部　管理栄養学科³, 名古屋大学医学部付属病院　先端医療・臨床研究支援センター⁴ Dept. Biochemistry, Nagoya University Graduate School of Medicine, Nagoya, Japan¹, Dept. Orthopedics, Nagoya University Graduate School of Medicine, Nagoya, Japan², Dept. Health and Nutrition, Yamanashi Gakuin University, Kofu, Japan³, Center for Advanced Medicine and Clinical Research, Nagoya University Graduate School of Medicine, Nagoya, Japan⁴ Amyotrophic lateral sclerosis (ALS) is a motoneuron-degenerative disease, the pathogenesis of which requires both cell autonomous and non-cell autonomous processes. Although many important proteins and gene mutations involved in the pathogenesis have been identified, sugar chains have been poorly studied in this process, and their biological significance remains largely unknown. Here, we investigate the role of keratan sulfate (KS) in ALS pathogenesis. KS expression was observed in a subpopulation of microglia in SOD1G93A mice, and became detectable around motoneurons in the ventral horn during the presymptomatic phase. In addition, we have employed ALS model SOD1G93A mice and GlcNAc6ST-1-/- mice, which are KS-deficient in the central nervous system. Surprisingly, SOD1G93AGlcNAc6ST-1-/- mice exhibited a significantly shorter lifespan than SOD1G93A mice and an accelerated disease onset. This study suggests that KS plays an indispensable, suppressive role in the early phase pathogenesis of ALS.	P2-1-212 自然免疫TRIF経路遮断によってALS疾患の進行が顕著に加速する Elimination of innate immune system adaptor TRIF significantly accelerates disease progression of ALS mice ○小峯起¹, 藤森典子¹, 山下博史², 森脇康博³, 三澤日出巳³, 山中宏二¹ ○Okiru Komine¹, Noriko Fujimori¹, Hirofumi Yamashita², Yasuhiro Moriwaki³, Hidemi Misawa³, Koji Yamanaka¹ 理研・BSI・運動ニューロン¹, 京都大院・医・臨床神経², 慶應大・薬・薬理³ Lab MND, BSI, RIKEN, Saitama¹, Dept Neurol, Kyoto Univ, Kyoto², Dept Pharmacol, Keio Univ, Tokyo³ Recent works demonstrated that adaptive immune system is involved in the motor neuron disease process. However, the role of innate immune system in motor neuron disease was not fully investigated. To test the role of innate immunity in the pathogenesis of ALS, SOD1^G93A ALS model mice were mated with MyD88 and TRIF (TIR domain-containing adaptor inducing IFNβ) deficient mice. MyD88 and TRIF are the essential adaptor proteins for Toll-like receptor mediated signaling pathway. As compared with SOD1^G93A mice, MyD88/TRIF double-deficient and TRIF-deficient SOD1^G93A mice exhibited the substantially shorter survival times with accelerated disease progression. The disease duration was shortened by 50% in TRIF-deficient SOD1^G93A mice. In contrast, elimination of MyD88 in SOD1^G93A mice showed marginal effect in survival time. In addition, the expression levels of pro-inflammatory chemokines, CCL5 and CXCL-10 were significantly suppressed in the spinal cord of TRIF-deficient SOD1^G93A mice, as compared with SOD1^G93A mice. To determine the cell type in which TRIF-dependent pathway contributes to the production of these chemokines, we examined the expression of chemokines in LPS-stimulated primary microglia or astrocyte derived from TRIF-deficient or MyD88-deficient mice. TRIF-dependent induction of these chemokines was observed only in LPS-stimulated microglia. Moreover, we found that infiltration of cytotoxic T-lymphocytes, natural killer T-lymphocytes and natural killer cells were significantly decreased in the spinal cord of symptomatic TRIF-deficient SOD1^G93A mice. These results suggest that the basal level of TRIF-dependent innate immune activation of microglia is beneficial to slow disease progression of ALS models through the maintenance of pro-inflammatory chemokines and the infiltration of the immune cells to the spinal cords. The detailed analyses to clarify the role of these infiltrating immune cells are underway.
P2-1-213 中枢神経系各細胞リニエージにおけるFUSのRNA代謝調節の特徴 -ALS/FTLDの病態理解に向けて- Comparison of FUS-regulated transcriptome in four primary cell lineages in the central nervous system reveals cell-specific regulations in association with ALS/FTLD ○藤岡祐介¹, 石垣診祐¹, 増田章男², 井口洋平¹, 勝野雅央¹, 大野欽司², 祖父江元¹ ○Yusuke Fujioka¹, Shinsuke Ishigaki¹, Akio Masuda², Yohei Iguchi¹, Masahisa Katsuno¹, Kinji Ohno², Gen Sobue¹ 名古屋大学大学院医学系研究科　神経内科学¹, 名古屋大学大学院医学系研究科　神経遺伝情報学² Dept Neurol, Nagoya University Graduate School of Medicine¹, Dev Neurogenetics, Nagoya University Graduate School of Medicine² FUS is a causative gene for familial amyotrophic lateral sclerosis (ALS) and pathologically links to sporadic ALS and frontotemporal lobar degeneration (FTLD). To clarify RNA metabolism cascade regulated by FUS in ALS/FTLD, we compared the FUS-regulated profiles of gene expression and alternative splicing among different primary cells in the central nervous system. More numbers of genes were differentially regulated by FUS in motor neurons, cortical neurons, and glial cells than in cerebellar neurons. The profiles of FUS-mediated alternative splicing between cortical neurons and motor neurons were quite similar but not with glial cells or cerebellar neurons, whereas the FUS-mediated gene expression profiles were similar among cortical neurons, motor neurons, and glial cells. FUS-mediated regulations of alternative splicing and gene expression are likely to dictate vulnerability of cells and cellular response, respectively. Some neurological diseases-associated genes including Mapt, Stx1a, and Fmr1 were identified to be regulated by FUS. Our results indicated that transcriptome profiles regulated by FUS were relevant to cell vulnerability in FUS-associated ALS/FTLD. Identified RNA targets for FUS could be therapeutic targets for ALS/FTLD.	P2-1-214 脱ユビキチン化酵素JosD1の機能的役割 Functional roles of the deubiquitinating enzyme JosD1 ○関貴弘^1,2, , 酒井規雄¹ ○Takahiro Seki^1,2, Sokol V. Todi^2,3, Norio Sakai¹, Henry L. Paulson² 広島大院・医歯薬保健・神経薬理¹, ミシガン大・神経内科², ウェイン州立大・医・薬理³ Dept Mol Pharmacol Neurosci, Grad Sch Biomed Sci, Hiroshima Univ, Hiroshima, Japan¹, Dept Neurol, Univ of Michigan, Ann Arbor, USA², Dept Pharmacol, Wayne State Univ Sch Med, Detroit, USA³ Ataxin-3 (ATXN3) is a deubiquitinating enzyme (DUB) that, when mutated to contain an expanded polyglutamine tract, causes the neurodegenerative disease Spinocerebellar Ataxia 3 (SCA3). The functional roles of ATXN3 and the relationship between its DUB activity and SCA3 pathogenesis, however, remain unknown. Josephin domain containing proteins 1 and 2 (JosD1 and JosD2) are two orthologues of ATXN3 that possess high similarity to the catalytic domain of ATXN3. Here, we examined the DUB activity and functional roles of JosD1 and JosD2. An in vitro DUB assay employing recombinant proteins revealed that JosD2 has higher DUB activity than JosD1. Ubiquitination of JosD1 increased its DUB activity, indicating that DUB activity of JosD1 is regulated by its ubiquitination, similar to ATXN3. When expressed in 293 cells, V5-tagged JosD1 strongly localized to plasma membrane, while JosD2-V5 existed diffusely in cytoplasm. Because of its membrane localization, we speculated that JosD1 participates in membrane-dependent events such as cell motility and endocytosis. Employing time lapse imaging in transfected 293 cells, we observed that GFP-JosD1 enhanced membrane dynamics and cell motility. These effects were also induced by a catalytically inactive form of JosD1, suggesting that JosD1 may regulate membrane dynamics and cell motility independent of its DUB activity. We also tested whether JosD1 affects endocytosis through the uptake of endocytic markers. Indeed, overexpression of JosD1 significantly increased the uptake of Lucifer yellow and 70kDa dextran, two commonly used markers of macropinocytosis, in a DUB activity-dependent manner. Taken together, the current results establish that JosD1 is a membrane-associated DUB whose activity is regulated by ubiquitination and that regulates membrane dynamics, cell motility and endocytosis. Because of the high similarity between JosD1 and ATXN3, these functions conceivably may be regulated by ATXN3 and relate to pathogenesis of SCA3.

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