e ポスター

e ポスター 10. 神経疾患の分子基盤 e Poster 10. Molecular basis of neurological disease

2020/9/11　14:00～15:00　オンデマンドB-1 P2-27 老化に伴うAβ病理形成メカニズムの解析：オートファジーの低下はエクソソームを介して細胞外へのAβ排出を促進する Downregulation of autophagy enhances exosome secretion to ameliorate intracellular accumulation of Aβ caused by endocytic disturbance. 木村展之¹、鯉沼真吾^1,2、下澤律浩³、保富康宏³ 1. 国立長寿医療研究センター、2. 東京理科大学、3. 医薬基盤・健康・栄養研究所 Nobuyuki Kimura¹, Shingo Koinuma^1,2, Nobuhiro Shimozawa³, Yasuhiro Yasutomi³ 1. National Center for Geriatrics and Gerontology, 2. Tokyo University of Science, 3. National Institutes of Biomedical Innovation, Health and Nutrition Abnormal accumulation of β-amyloid protein (Aβ) in brain is the key factor for Alzheimer's disease (AD) pathogenesis. We have previously shown that aging attenuates the interaction of the microtubule-based motor protein dynein with dynactin, and dynein dysfunction disrupts endosome trafficking, resulting in intracellular accumulation of Aβ. However, it remains unclear how intracellular accumulation of Ab leads to extracellular deposition, so-called amyloid plaque. Recent findings showed that autophagy is involved in the degradation of Aβ and that the expression levels of autophagy-related genes are reduced in AD patient brains. Thus, in the present study, we aim to clarify the relationship of autophagy with age-related Aβ pathology. In cynomolgus monkey brains, the levels of autophagy-related proteins, such as ATG5 and LC3, decreased in an age-dependent manner in cynomolgus monkey brains. To investigate how autophagy affects intracellular/extracellular Aβ levels, we examined RNAi studies combined with chemical treatments in Neuro2a cells. Surprisingly, rapamycin (Rm) treatment strongly augments dynein knockdown-induced intracellular accumulation of Aβ and decreased extracellular Aβ levels. Previous studies showed that Aβ is released extracellularly via exosome. Evidently, Rm treatment strongly disrupted exosome secretion. On the other hand, ATG5 knockdown enhanced exosome secretion and ameliorated intracellular accumulation of Aβ caused by dynein knockdown. Finally, we confirmed that the amount of Aβ significantly increased in exosomes derived from brain parenchyma of aged monkey brains, and we confirmed the localization of exosome marker proteins in amyloid plaques. These findings suggest that autophagy can affect extracellular release of Aβ via exosomes, and that the downregulation of autophagy-related proteins in aged monkey brains and even in AD brains may be a compensatory response to reduce intracellular accumulation of Aβ via enhanced exosome secretion.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-28 遺伝子改変マウスを用いたアルツハイマー型認知症の血液トランスクリプトームバイオマーカーの検索 Identifying blood transcriptome biomarkers of Alzheimer's disease using transgenic mice. 伊賀淳一¹、越智紳一郎¹、舟橋裕¹、吉野祐太¹、山崎聖広¹、久門啓志¹、森大晃¹、尾崎優樹¹、森崇明¹、上野修一¹ 1. 愛媛大学 Junichi Iga¹, Shinichiro Ochi¹, Yu Funahashi¹, Yuta Yoshino¹, Kiyohiro Yamazaki¹, Hiroshi Kumon¹, Hiroaki Mori¹, Yuki Ozaki¹, Takaaki Mori¹, Shu-ichi Ueno¹ 1. Ehime Univ. Background: Testing for pathological biomarkers of Alzheimer's disease (AD) such as amyloid beta and tau is time-consuming, expensive, and invasive. Here we used 3xTg-AD mice to identify and validate putative novel blood transcriptome biomarkers of AD that could potentially be identified in the blood of patients. Methods: mRNA was extracted from the blood and hippocampus of 3xTg-AD and control mice at different ages and used for microarray analysis. Results: The networks and functional analyses revealed that differentially expressed genes modulated the immune and neuroinflammation system. We also found five novel gene transcripts (Cdkn2a, Apobec3, Magi2, Parp3, Cass4) that were significantly increased with age and whose expression was correlated between the blood and hippocampus only in AD mice. We further assessed previously identified candidate biomarker genes. Trem1 and Trem2 were significantly increased with age in both blood and brain. Decreased Tomm40 and increased Pink1 mRNA levels were observed in mouse blood. Expression of Snca and Apoe mRNA in mouse blood and brain showed similar changes as those in human AD blood. Conclusion: Our results demonstrated that the immune and neuroinflammation system was involved in the pathophysiology of aging and AD and blood transcriptome may be useful as biomarkers of AD.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-29 タウオパチー依存的なタウのリン酸化 Disease-dependent phosphorylation of tau among different Tauopathies Samimi Nastaran ^1,2,3、Sharma Govinda ¹、木村妙子^1,5、村山繁雄⁴、長谷川成人⁵、 Farjam Mojtaba³、Koorosh Shahpasand²、安藤香奈絵¹、久永眞市¹ 1. 東京都立大学、2. Royan Institute for Stem Cell Biology and Technology、3. Fasa University of Medical Sciences、4. 東京都健康長寿医療センター研究所、5. 東京都医学総合研究所 Nastaran Samimi^1,2,3, Govinda Sharma¹, Taeko Kimura^1,5, Shigeo Murayama⁴, Masato Hasegawa⁵, Mojtaba Farjam³, Koorosh Shahpasand², Kanae Ando¹, Shin-ichi Hisanaga¹ 1. Tokyo Metropolitan University, 2. Royan Institute for Stem Cell Biology and Technology, 3. Fasa University of Medical Sciences, 4. Tokyo Metropolitan Institute of Gerontology, 5. Tokyo Metropolitan Institute of Medical Science Tauopathies are a spectrum of neurodegenerative diseases characterized by accumulation of pathological tau protein. While tau's MT-assembly activity is regulated physiologically by phosphorylation, it's hyperphosphorylation is proposed to play a critical role in tauopathy development. However, there are no reports addressing the detailed phosphorylation states among tauopathies. In this study, we aimed to evaluate the phosphorylation states of tau among different tauopathies including Cortico-Basal Degeneration (CBD), Pick's disease (Pick), Argyrophilic Grain Dementia (AGD), Alzheimer's disease (AD), comparing with those of healthy individuals. At first, we examined tau phosphorylation by Phos-tag SDS-PAGE, which separates phosphorylated proteins depending on the amount and sites. The banding profiles of tau were different among tauopathies, suggested the different phosphorylation in different tauopathies. Then, we analyzed phosphorylation sites using a panel of anti-phosphorylation site-specific antibodies. Immunoblots showed significantly higher phosphorylation at Ser202 in Pick and AD patients. AGD patients showed stronger reactions with anti-PS396. Also, tau in AD brains were more phosphorylated at Thr231 and Ser235. Further, AGD and AD brains showed strong reactions with anti-cis P-tau, a pathological phosphorylation of Thr231. We confirmed the decreased amount of Pin1 Cis-Trans isomerase in AGD and AD brains, compared to other samples. These results indicate the disease-specific phosphorylation patterns of tau among tauopathies. In particular, high levels of cis p-tau in AGD and AD patients suggest similar tau pathology in these diseases. Altogether, these data may be beneficial for finding the molecular mechanism and diagnostic approaches for different tauopathies.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-30* 神経回路形成因子LOTUSによるアミロイドβタンパク質受容体PirBの制御 Regulation of an amyloid β protein receptor PirB by a neural circuit formation factor LOTUS 川口祐生¹、栗原裕司¹、松林潤平¹、川上裕¹、竹居光太郎¹ 1. 横浜市立大学生体機能医科学研究室　 Yuuki Kawaguchi¹, Yuji Kurihara¹, Junpei Matsubayashi¹, Yutaka Kawakami¹, Kohtaro Takei¹ 1. Molecular Medical Bioscience Laboratory, Yokohama City University Graduate School of Medical Life Science It has been recently reported that Nogo receptor type 1 (NgR1) and Paired immunoglobulin-like receptor B (PirB) act as a receptor of amyloid beta (Aβ) protein, known as a main risk factor of Alzheimer's disease (AD). We identified lateral olfactory tract usher substance (LOTUS) as an endogenous antagonist of both NgR1 and PirB. In this study, we examined whether LOTUS is involved in the regulation of Aβ binding to NgR1 or PirB and that of Aβ-induced decrease of synaptic density in hippocampal neurons. We first confirmed that Aβ oligomer formed by the polymerization of Aβ monomers bound to PirB. We next examined whether LOTUS inhibits the binding of Aβ to PirB in COS7 cells. The membrane-bound type of LOTUS inhibited partially (about 20% decrease) the binding of Aβ to PirB, whereas the soluble form of LOTUS (sLOTUS) did not. We then examined the effect of sLOTUS on Aβ-induced decrease of synaptic density in cultured hippocampal neurons. Though the synaptic density is reduced by Aβ treatment, this reduction was partially suppressed by sLOTUS treatment. The data suggest that LOTUS may suppress Aβ-induced actions such as inhibition of synaptic plasticity, thereby alleviating Aβ-induced pathogenesis. This inhibitory effect by LOTUS may lead to the idea that LOTUS may be a possible therapeutic agent for the progression of AD pathology.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-31 神経細胞のゴルジストレス誘導により引き起こされるAβペプチド合成の変化およびCaspase3依存性アポトーシスに対するケミカルシャペロンPBAの効果 Effect of chemical chaperone PBA on Golgi stress induced changes in Aβ peptide production and caspase3-dependent apoptosis in neuronal cells 須賀圭^1,2、山本幸子¹、寺尾安生²、赤川公朗²、丑丸真¹ 1. 杏林大学医学部化学教室、2. 杏林大学医学部病態生理学教室 Kei Suga^1,2, Sachiko Yamamoto¹, Yasuo Terao², Kimio Akagawa², Makoto Ushimaru¹ 1. Dept. of Chem., Kyorin Univ. Sch. of Med., 2. Dept. of Med. Physiol., Kyorin Univ. Sch. of Med. Involvement of the dysfunction of neuronal cells caused by Golgi stress in neurodegenerative diseases such as Alzheimer's disease (AD) is poorly understood. We have been focusing on the function of ER-Golgi soluble N-ethylmaleimide-sensitive factor-attachment protein receptors (ER-Golgi SNAREs) in βAPP processing under cellular stress. We previously showed that ER stress upregulates de novo synthesis of ER-Golgi SNAREs Syntaxin5 (Syx5), and the reduction of β-amyloid peptide (Aβ peptide) in neuronal cells (Exp. Cell Res., 2015, Neurosci. Lett., 2015). The reduction of Aβ secretion by ER stress was significantly suppressed by Syx5 knock down. Conversely, down regulation of Syx5 protein was observed upon apoptosis which was due to the degradation by activated caspase-3 (Data in Brief, 2015, 2016). In addition, we reported that a chemical chaperone 4-phenylbutyrate (PBA) showed alleviation of caspase3-dependent apoptosis induced by ER stress. Last year, we reported that multiple Golgi stress inducers also promote upregulation of ER-Golgi SNAREs Syx5, reduction of Aβ peptide secretion, and the caspase3-dependent apoptosis. However, we did not know whether PBA is also effective to cells treated with Golgi stress inducers. In order to examine the protective mechanism of PBA on the changes in the processing of βAPP, the amount of secreted Aβ, intracellular Aβ, and the ratio of Aβ peptides, we performed time lapse imaging, western blotting, and sandwich ELISA analyses in Golgi stress-induced neuronal cells. We will present the data for the effect of chemical chaperone PBA on the production of Aβ peptides, and the activation of caspase3-dependent apoptosis under Golgi stress condition in neuronal cells and would like to discuss the results.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-32* 新規環状ペプチドによるGM1含有脂質膜上で誘起されたアミロイドβ線維化の阻害 Inhibition of amyloid β assembly induced on GM1-containing lipid membrane by novel cyclic peptides 宮本恵里花¹、中井真子¹、西原昌哉¹、松原輝彦¹、佐藤智典¹ 1. 慶應義塾大学理工学部 Erika Miyamoto¹, Mako Nakai¹, Masaya Nishihara¹, Teruhiko Matsubara¹, Toshinori Sato¹ 1. Faculty of Sci. and Tech., Keio University Alzheimer's disease (AD) is a neurodegenerative disease and the most common type of dementia associated with loss of memory and cognitive dysfunction. One of the characteristics found in the patient's brain is senile plaques formed by the deposition of amyloid β protein (Aβ). It has been reported that toxic Aβ assemblies are induced by the binding of Aβ to ganglioside cluster in neuronal membranes. Although this mechanism has been investigated a lot so far, no AD therapeutic drug has been developed based on the mechanism. Therefore, we focused on the ganglioside cluster that has a potential for therapeutic target against AD. In present study, we investigated a GM1 cluster-binding peptide identified by a phage display method as an inhibitor of Aβ assembly. However, peptide has low metabolic stability and therefore need to be restricted its conformational space by cross-linking. We designed several cyclic peptides with intramolecular cross-link at different positions and evaluated its inhibitory activity for Aβ fibril formation. A GM1-containing lipid bilayer (GM1/sphingomyelin/cholesterol) was prepared using a Langmuir trough and the assembly of Aβ was evaluated by atomic force microscopy (AFM). Our results indicate that the cyclic peptides have affinity for GM1 cluster, and GM1-induced Aβ fibrils decreased in the presence of the GM1 cluster-binding cyclic peptides. These results suggest the GM1 cluster-binding cyclic peptides are available for a novel therapeutic candidate for AD.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-33* アルツハイマー病のin vitro細胞死モデルの最適化に向けたpH click Aβによる凝集体依存的な細胞毒性の解析 Analysis of aggregation-dependent cytotoxicity by pH click Aβ for the optimization of in vitro cell death model of Alzheimer's disease. 福田愛菜¹、西村周泰¹、高田和幸¹ 1. 京都薬科大学統合薬科学 Aina Fukuda¹, Kaneyasu Nishimura¹, Kazuyuki Takata¹ 1. Divi. Integ. Pharm. Sci., Kyoto Pharm. Univ. Alzheimer's disease (AD) is a representative neurodegenerative disease, and the pathological feature is accumulation of amyloid-β (Aβ) in brains. Aβ is easily prone to aggregate so that it forms a variety of multimeric complex from monomer to fibrils. Aβ oligomers are produced in the process of Aβ assembly and indicate stronger neurotoxicity than monomer and fibrils. Thus, Aβ oligomers are recognized as key molecules initiating the neurodegenerative process in AD (Aβ oligomer hypothesis). Since Aβ oligomers exist only transiently and forms fibrils rapidly, there is no stable cell death model caused by Aβ oligomers, and the mechanism of Aβ oligomer-induced neurodegeneration is still unknown. To overcome this issue, we focused on 26-O-acyl Aβ_1-42 (pH click Aβ), which is easily controlled its aggregation by pH (i.e. start to aggregate at neutral condition and stop at acidic condition). In the present study, we examined pH click Aβ-induced cell death and analyzed the relevance of cell toxicity and Aβ aggregates formations. The pH click Aβ solved with trifluoroacetic acid were incubated at 37℃ under neutral pH condition and Aβ aggregations were analyzed by Western blot analysis. The pH click Aβ were also applied to human SH-SY5Y neuroblastoma cells, and cell viability was analyzed by WST-8 assay. Aβ monomer and oligomers (from dimer to pentamer) existed at the beginning of the incubation and were gradually decreased, while Aβ fibrils were inversely formed. Although pH click Aβ significantly induced cell death, it was attenuated along with decreasing Aβ oligomers. Thus, we have successfully established a stable in vitro cell death model using pH click Aβ which may reflect on Aβ oligomer hypothesis. Application of human neuronal cells, such as human induced pluripotent stem cell-derived cholinergic neurons, may dramatically increase the significance of this pH click Aβ induced cell death model as a tool for the clarification of AD pathophysiology and screening of anti-AD drugs.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-34 C9orf72リピート延長変異FTLD/ALSにおけるRNA代謝障害 Dysregulated repeat RNA metabolism in C9orf72-FTLD/ALS 森康治¹、河邉有哉¹、後藤志帆¹、山下智子¹、池田学¹ 1. 大阪大学精神医学 Kohji Mori¹, Yuya Kawabe¹, Shiho Gotoh¹, Tomoko Yamashita¹, Manabu Ikeda¹ 1. Psychiatry, Osaka University An intronic GGGGCC (G₄C₂) repeat expansion in C9orf72 is a leading genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The hexanucleotide DNA repeat is bidirectionally transcribed into repeat RNA accumulating within RNA foci. We revealed the repeat RNA is even translated into five distinct proteins in the absence of the canonical initiation codon (AUG) into dipeptide-repeat protein (DPR)s with tandem dipeptide repeat motifs. Since the identification of DPR, cytotoxic properties of DPR has been revealed using multiple disease models. DPR could be primary driving force for neurodegeneration in C9orf72-FTLD/ALS. Repeat-dependent toxicity in C9orf72 may affect RNA splicing, nuclear import machinery, status/function of membraneless organelles including RNP granules, stress granules and nucleolus. HnRNPA3 is a nucleo-cytoplasm shuttling heterogeneous nuclear ribonucleoprotein (hnRNP) that we previously found specifically binds to the G₄C₂ repeat RNA. We revealed a reduction of hnRNPA3 leads to accumulation of repeat RNA and RNA foci as well as deposition of DPR proteins. Moreover, we recently found a nucleolar RNA degradation machinery confers repeat RNA metabolism. In summary, we identified endogenous modulators of the C9orf72 repeat RNA levels. We postulate dysregulated repeat RNA metabolism may underlie prominent repeat RNA and DPR depositions in C9orf72-FTLD/ALS.

2020/9/11　14:00～15:00　オンデマンドB-1 P2-35 C9orf72 FTLD/ALS細胞モデルにおけるリピート関連ATG非依存性翻訳の調整 Regulating repeat associated non-ATG translation in a cellular model of C9orf72 FTLD/ALS 後藤志帆¹、森康治¹、河邉有哉¹、山下智子¹、近江翼¹、池田学¹ 1. 大阪大学精神医学 Shiho GOTOH¹, Kohji Mori¹, Yuya Kawabe ¹, Tomoko Yamashita¹, Tsubasa Omi¹, Manabu Ikeda¹ 1. Psychiatry, Osaka Univ. Though clinical phenotypes of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS) widely differ, an abnormally extended GGGGCC repeat in noncoding region of C9orf72 is a common cause of genetically inherited version of both FTLD and ALS (C9 FTLD/ALS). The GGGGCC repeat could be transcribed in both sense and antisense directions. Then the transcribed repeat RNA is translated into accumulating dipeptide repeat proteins (DPR) via repeat-associated non-ATG (RAN) translation. DPR accumulate in the brain of C9 FTLD/ALS patients. Since DPR are even considered to be neurotoxic, selective inhibition of RAN translation may provide a venue for novel therapeutics. However, precise mechanism of RAN translation is still obscure. In the present study, we investigated how RAN translation is regulated in a cellular model of C9 FTLD/ALS. First, we knocked down several candidate proteins if they affect the efficiency of RAN translation. By doing so, we identified one promising protein which might regulate RAN translation (Here we designate it as translational regulator 1 (TR1)). With TR1 knockdown, cellular expression level of a DPR was decreased. Conversely in TR1 overexpressing cells, the expression level of the DPR was increased. These results suggest that TR1 regulates the expression level of DPR. This was not due to the increase in repeat RNA expression, since no additional repeat RNA accumulation was observed upon TR1 overexpression. Importantly, inactive mutant TR1 failed to increase DPR expression. Furthermore, surface sensing of translation (SUnSET) assay revealed rather selective increase of RAN translation over conventional AUG-dependent translation on TR1 overexpression. Collectively, we will present evidence that TR1 selectively regulates RAN translation. TR1 inhibition might offer a novel therapeutic strategy for C9-FTLD/ALS.		2020/9/11　14:00～15:00　オンデマンドB-1 P2-36 Praja 1 RING-finger E3 ubiquitin ligaseによるTDP-43細胞質凝集体の形成抑制 Praja 1 RING-finger E3 ubiquitin ligase suppresses neuronal cytoplasmic TDP-43 aggregate formation 渡部和彦^1,2、加藤洋一郎²、佐久間美帆³、村田麻喜子¹、新井田素子²、竹村太郎⁴、花方信孝⁴、他田真理⁵、柿田明美⁵、柴田亮行² 1. 杏林大学保健学部臨床検査技術学科神経病理学、2. 東京女子医科大学病理学講座病態神経科学分野、3. 東京女子医科大学医学部医学科、4. 物質・材料研究機構技術開発・共用部門、5. 新潟大学脳研究所病理学分野 Kazuhiko Watabe^1,2, Yoichiro Kato², Miho Sakuma³, Makiko Murata¹, Motoko Niida², Taro Takemura⁴, Nobutaka Hanagata⁴, Mari Tada⁵, Akiyoshi Kakita⁵, Noriyuki Shibata² 1. Dept. Medical Technology, Kyorin Univ. Faculty of Health Sciences, 2. Dept. Pathology, Tokyo Woman's Med. Uniiv., 3. Sch. Med., Tokyo Woman's Med. Uniiv., 4. Research Network and Facility Services Division, National Inst. Materials Science, 5. Dept. Pathology, Brain Research Inst., Niigata Univ. Transactivation response DNA-binding protein-43 (TDP-43) is a main constituent of cytoplasmic aggregates in neuronal and glial cells in cases of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We have previously demonstrated neuronal cytoplasmic aggregate formation induced by recombinant adenoviruses expressing human wild type (WT) and C-terminal fragment (CTF) TDP-43 under the condition of proteasome inhibition. The formation of the adenoviral TDP-43 aggregates was markedly suppressed by co-infection of an adenovirus expressing heat shock transcription factor 1 (HSF1), a master regulator of heat shock response. In the present study, we performed DNA microarray analysis and selected several candidate molecules locating downstream of HSF1 that counteract TDP-43 aggregate formation. In these, we identified Praja 1 RING-finger E3 ubiquitin ligase (PJA1) as a suppressor of phosphorylation and aggregate formation of TDP-43. Co-immunoprecipitation assay revealed that PJA1 binds to CTF TDP-43 and E2 conjugating enzyme UBE2E3. PJA1 also suppressed formation of adenoviral cytoplasmic phosphorylated TDP-43 aggregates in mouse facial motor neurons in vivo. Furthermore, phosphorylated TDP-43 aggregates were demonstrated in PJA1-immunoreactive human ALS motor neurons. These results indicate that PJA1 is one of the principal E3 ubiquitin ligase for TDP-43 to counteract its aggregation propensity and could be a potential therapeutic target for ALS and FTLD.