○千崎 健佑、岡田 陽子、明地 雄司、武井 聡子、松本 清香、
三浦 史郎、越智 雅之、越智 博文、伊賀瀬道也、大八木保政 愛媛大学大学院医学系研究科 脳神経内科・老年医学講座
【目的】血管拡張反応を利用した血管内皮機能の解析は、循環器系疾患だけでなく、
認知症・神経変性疾患や自己免疫性神経疾患などでも近年報告が増加している。
今回我々は、脳神経疾患患者を対象として、超音波検査を用いた血管内皮機能評
価を行い、疾患との関連を検討した。【方法】対象は当院に入院・外来加療中の患
者で、研究に同意が得られた72例。TCDを用いた息こらえ試験Breath Holding Index （BHI）と上腕動脈のFlow Mediated Dilatation （FMD）の2種類の超音波に よる血管内皮機能検査を施行した。【結果】平均年齢は58.1歳（22-86歳）、男性34例、
女性38例であった。今回の対象全体における平均値はBHI 0.85±0.6、収縮期％
FMD 4.3±2.8であった。年齢とBHIは有意な逆相関があり（R2=0.08, p=0.022）、
FMDでも年齢との逆相関傾向が見られた（R2=0.09, p=0.051）。過去に報告されて いる正常値を用いると、今回の検討例における血管内皮機能障害の頻度はBHI 0.7 未満；31例43.1％、FMD 6％未満；35例48.6％であった。基礎疾患を認知症（主に アルツハイマー型認知症AD）、変性疾患、炎症性疾患、その他に分類し、血管 内皮機能との関係を検討したところ、FMD低下群では認知症が多い傾向を認め た（BHI正常群8例19％ vs 低下群5例17％、％ FMD正常群1例13％ vs 低下群8例
23％）。【結論】ADの発症や病態進行にはNO産生低下による脳微小血管障害の関与
が報告されている。今回の我々の検討の結果から、ADにおける血管内皮障害は、
BHIよりも主にNOに対する血管拡張反応を示すFMDに反映されやすい可能性が 示唆された。脳神経疾患において両解析を比較・検討した報告はまだなく、文献 的考察を加えて報告する。
O-38-9
軽度認知機能障害（MCI）患者における脳小血管病ス コアと認知機能の検討○松田 佳奈1,2、伊井裕一郎1、上田有紀人2、田部井賢一1,3、
石川 英洋1、新堂 晃大1、松浦 慶太1、吉丸 公子1,3、谷口 彰3、 加藤奈津子1,3、田村 麻子1、伊藤 愛1、佐藤 正之3、冨本 秀和1
1 三重大学大学院医学系研究科神経病態内科学、
2 三重大学医学部附属病院リハビリテーション部、
3 三重大学大学院医学系研究科認知症医療学
【目的】脳小血管病 （SVD） の重症度を評価するスコアとして，高血圧性SVDス コア （以下 hypertensive vasculopathy; HV-SVDスコア）と脳アミロイド血管症
（CAA）を反映するスコア （以下 CAA-SVDスコア）がある。前者は知的機能と相 関するとの報告はあるが，後者に関する報告はない。今回，軽度認知機能障害
（MCI） 患者においてCAA-SVDスコアと認知機能との関連について検討した。【方 法】対象は，2017年2月～2019年7月に当院でMCIと診断され，脳MRI （3T-MRIで FLAIR，DIR，SWI，T1，T2） の撮像が可能だった42名 （75.3±9.12歳，男性23 名，女性19名） で，知的機能・記憶・前頭葉機能・構成などの認知機能を評価した。
CAA-SVDスコアは，Charidimou Aら （JAMA Neurol 2016） に基づき，脳葉型 脳微小出血，脳表ヘモジデリン沈着症，半卵円中心の血管周囲腔拡大および白質 病変の各スコアの総計を0～6の7段階で算出し，認知機能の各指標を独立変数と して重回帰分析を行った。また，白質病変定量解析ソフトFUSIONで白質病変を 定量し後方優位に1点，CAAに起因する皮質微小梗塞 （Ishikawa H, in press）があ
れば1点を加えたスコアと比較した。【結果】CAA-SVDスコアと認知機能に相関を
認めた （r=0.902，p=0.013）。CAA-SVDスコアに白質病変後方優位，CMI （＋） を 加えると若干回帰式の予測精度が高くなった （r=0.908，p=0.010）。決定係数R2は 0.813から0.824と向上した。CAA-SVDスコアは知的機能，記憶，構成能力と相関
した。【結論】CAA-SVDスコアはMCI患者の知的機能のみならず，記憶，構成能力
とも相関する。
O-38-10
Cingulateislandsign（CIS）指標における年齢層別 cut-offvalueの検討○ 内田 大達、木村 成志、麻生 泰弘、石橋 正人、軸丸 美香、
松原 悦朗
大分大学医学部神経内科
【目的】レビー小体型認知症（DLB）のFDG-PETでは、後頭葉の糖代謝低下に加えて 後部帯状回の糖代謝が保たれる。この所見は、Cingulate island sign:CISと呼ばれ、
アルツハイマー型認知症（AD）との鑑別診断に有用である。近年、脳血流SPECT 画像を用いたCIScoreの解析法が報告され、実臨床においても活用させている。
我々は、DLBとADの鑑別における年齢層別のCIScore cut-off 値の変化を検討す る。【方法】DLBの診断基準を満たし、MIBG心筋シンチH/Mの後期像が2.1以下で ある54例（男：女=25：29，平均年齢 76.7 ± 7.6 歳）、およびNINCDS-ADRDA 診 断基準を満たし、脳血流SPECTでADの関心領域に血流低下を認める120例（男：
女=62：58，平均年齢 73.7 ± 9.1 歳）を対象とした。DLBで脳血流が低下する後頭 葉を中心とした領域とADに比較してDLBで血流が保たれる後部帯状回を関心領 域に設定し、z値の比をCIS指標とした。DLBとAD症例を70歳未満、70-79歳、80 歳以上に分けROC解析によりADとDLBの鑑別診断において最適なCIS指標のcut-off値、感度、特異度、正診率を算出した。【結果】70歳未満のcut-off値は、0.250で あり、正診率80.5%であった。70-79歳、cut-off値は、0.265であり、正診率78.5%で あった。80歳以上のcut-off値は、0.305であり、正診率73.3%であった。【結論】CIS 指標は、ADとDLBの鑑別診断に有用であるが、高齢になるほど鑑別に最適なcut-off値が高値となり、正診率が低下する。この原因として合併病理の影響が考えら れる。
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Perampanelimprovesseizureviathephosphorylation ofGluA1intheDRPLAtransgenicmice○TomokoToyota1,ZheHuang2,KazumasaOkada1,ToshiyaSato3, ShojiTsuji4,5,HiroakiAdachi1
1 Department of Neurology, University of Occupational and Environmental Health School of Medicine, Japan, 2 Shanghai TCM-integrated Hospital,
3 Department of Laboratory Animal Science, Kitasato University School of Medicine, 4 Department of Neurology, International University of Health and Welfare, 5 Department of Molecular Neurology, University of Tokyo Graduate School of Medicine
Objective: The juvenile-onset dentatorubural-pallidoluysian atrophy （DRPLA） presents progressive myoclonic epilepsy （PME）: ataxia, myoclonus, seizures, and progressive intellectual deterioration. DRPLA is caused by an expanded polyglutamine tract within the atrophin-1 and an autosomal dominant fatal disease. There are no disease-modifying therapies at present. Recently, perampanel （PER）, a selective competitive a-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid （AMPA） receptor antagonist, was reported to be effective for PME. In this study, we investigated the effects of PER to the Q113 and Q129 DRPLA transgenic model mice that present PME. Methods: PER was administrated to Q113 and Q129 DRPLA transgenic mice 2.5 mg/kg （Q113, n=6; Q129, n=11）, 5 mg/kg （Q113, n=6, Q129, n=15）, or 10mg/kg （Q113, n=8; Q129, n=10） respectively everyday via oral gavage from 6-week-old （Q113） and 4-week-old （Q129）. The control DRPLA transgenic mice （Q113, n=7; Q129, n=17） were received the same volume methyl cellulose solution. We observed myoclonus and epilepsy with video. We analyzed motor function by rotarod task. Western blotting and immunohistochemical analysis of brain tissues were performed using antibodies against glutamate receptor 1 （GluA1） and phosphorylated GluA1. Results: PER was effective for myoclonus and seizure with significant difference （P<0.05）. In immunohistochemical and biological analysis, perampanel increased the phosphorylation of GluA1. Conclusions: The phosphorylation of GluA1 may contribute to improve myoclonus and seizure in the DRPLA transgenic mice.
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Calcium-bindingproteinsstimulatemicrogliato induceproinflammatorymediatorssimilartoALS○ShintaroHayashi1,2,RyoYamasaki1,KoichiOkamoto3,Jun-ichiKira1
1 Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Japan, 2 Department of Neurology, Gunma Rehabilitation Hospital, Japan, 3 Depatment of Neurology, Geriatrics Research Institute and Hospital
Objective:In amyotrophic lateral sclerosis （ALS） spinal cord, microglia/
macrophages in the anterolateral funiculi outside the corticospinal tracts （ALFoc）, not those in the motor system, were significantly involved in TDP-43 pathology of motor neuron （Hayashi S, 2018）. The present study aimed to clarify the cause of microglial activation in the ALFoc of ALS, with referring that calcium-binding protein （CaBP）-immunoreactive axons are abundantly present in the ALFoc of normal human spinal cord （Fournet N, 1984）. Method:Cultured microglia （1.65
×104 cells/well/100μl） were incubated by 2 kinds of CaBPs, calbindin （CB）
（cytoplasmic CaBP） and calreticulin （CALR） （endoplasmic reticulum CaBP）, both with 1μM, 1nM, and 1pM, and by culture medium （control）. Cytokines/
chemokines concentrations （CXCL1, IFN-gamma, IL-1beta, IL-10, IL-12, IL-6, MCP1, TNF-alpha） in the supernatants were measured by Immunoassay Panel at day 0 （prior to incubation）, 1, 2, and 3. At each day, microglial cells were fix by paraformaldehyde, and crystal violet stainings were performed. Results:Among the 8 cytokines/chemokines, only TNF-alpha was elevated at day 1 and continued to increase with CB incubation （1μM and 1 pM）. IL-6, MCP-1, and TNF-alpha were elevated by day 1 with incubation of CALR （1μM and 1 pM）.
Microglia had been alive during the day 0-3. Conclusions:This study first showed that CaBPs stimulate cultured microglia to induce proinflammatory cytokines/
chemokines. The elevated cytokine/chemokine profiles are similar to those in the cerebrospinal fluids of patients with ALS （Tateishi T, 2010）.
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HDAC10KOactivateschaperone-mediatedautophagy andacceleratesthedecompositionofitssubstrate○YoshitoNagano1,2,HitomiObayashi1,TetsuyaTakahashi1, TakahiroSeki3,ShigeruTanaka4,NorioSakai4,
MasayasuMatsumoto5,HirofumiMaruyama1
1 Department of Clinical Neuroscience and Therapeutics, Graduate School of Biomedical and Health Sciences, Hiroshima University, Japan, 2 Mitsubishi Tanabe Pharma, Japan, 3 Department of Chemico-Pharmacological Sciences, Graduate School of Pharmaceutical Sciences, Kumamoto University, 4 Department of Molecular and Pharmacological Neuroscience, Graduate School of Biomedical & Health Sciences, Hiroshima University, 5 Sakai City Medical Center
[Objective]Recent reports indicate that a dysregulation of chaperone-mediated autophagy（CMA） contributes to the onset or progression of Parkinson's disease（PD）. α-Synuclein（αS）, which is a major component of Lewy bodies（LBs） in PD, has been identified as a CMA substrate. Previously, we found that histone deacetylase（HDAC）10 is localized with αS in LBs. HDAC10 is a member of class IIb HDACs, but its functions are poorly characterized. Recent study has shown that HDAC10 deacetylates HSC70 which plays an important role in CMA. Accordingly, we hypothesize that HDAC10 participates in a CMA regulation and contributes to the pathogenesis of PD. The aim of this study is to clarify whether HDAC10 is involved in CMA.[Methods]We established HDAC10 knockout HeLa cell lines using CRISPR/Cas9 system.
Immunoblotting and RT-PCR were used to determine the expression levels of LAMP2A. To directly investigate the CMA activity at a single-cell level, we utilized the GAPDH-HaloTag（HT） indicator system, which could monitor the translocation of GAPDH, a well-known CMA substrate, from cytoplasm to lysosomes.
Pulse chase assay using GAPDH-HT was preformed to investigate the degradation rate of GAPDH.[Results]In HDAC10 knockout cells, LAMP2A protein and mRNA levels were increased and LAMP2A-positive lysosomes accumulated around the nucleus. GAPDH was delivered to LAMP2A-positive lysosomes and degraded in HDAC10 knockout cells more efficiently than in wild type cells.[Conclusions]HDAC10 participated in regulating CMA, and HDAC10 knockout activated CMA and accelerated degradation of a CMA substrate.
一 般 演 題 口 演
O-39-10
Quantumdot/nanogoldcolabelingfordoublecorrelative lightandelectronmicroscopyofhumanbrain○MihoUematsu1,2,3,4,KyoheiMikami5,AyakoNakamura1,6,7, KatsuikuHirokawa8,EijiroAdachi1,RyosukeTakahashi2, ToshikiUchihara1,6,7
1 Laboratory of Structural Neuropathology, Tokyo Metropolitan Institute of Medical Science, Japan, 2 Department of Neurology, Kyoto University Graduate School of Medicine, Japan, 3 Department of Immunology and Genomics, Osaka City University Graduate School of Medicine, Japan,
4 Division of Innate immune regulation, International Research and Development Center for Mucosal Vaccine, Institute of Medical Science, The University of Tokyo, Japan, 5 Center for basic technology research, Tokyo Metropolitan Institute of Medical Science, 6 Department of neurology, Nitobe-Memorial Nakano General Hospital, 7 Department of Neurology and Neurological Science, Tokyo Medical and Dental University, 8 Department of pathology, Nitobe-Memorial Nakano General Hospital
[Objective] Cadmium selenide （CdSe） quantum dot （QD） is a nanocrystal which is detectable both as a fluorescent signal by light microscopy （LM） and as an electron-dense particle by electron microscopy （EM）. It is a suitable immunolabeling probe for correlative light and electron microscopy （CLEM） of the postmortem formalin-fixed human brain section, as we have established previously. Fluoronanogold is another agent with similar dual visibility. Double CLEM by combining these probes would further expand the study of ultrastructural localization of immunolabeled proteins in the brain.
[Materials and Methods] Brain sections with neuronal inclusions and astrocytes were double-immunofluorolabeled using QDs and fluoronanogolds, respectively. The targeted structures preselected on LM were identified for EM preparation by landmarks that we placed on the sections, and subsequently subjected to EM observation to identify immunolabels on the ultrastructure. [Results] Ultrastructural visualization of the targeted structures which were corresponding to fluorescent images demonstrated the presence of the QDs and nanogolds on the immunolabeled filaments on EM. Specificity of both labels could be distinguished by energy dispersive X-ray spectroscopy. [Conclusions] This is the world's first research that established double CLEM of postmortem diseased brain sections using the combination of QDs and fluoronanogolds. This method would demonstrate how different molecules woven into complex three-dimensional structures are at work in situ.
O-40-1
Blood-brainbarrier-activationinmyelinoligodendrocyte glycoproteinantibodyassociateddisorders○FumitakaShimizu1,RyoOgawa2,ToshiyukiTakahashi2,
YukioTakeshita1,TatsuroMisu2,YasuteruSano1,ToshihikoMaeda1, IchiroNakashima3,KazuoFujihara4,TakashiKanda1
1 Department of Neurology and Clinical Neuroscience, Yamaguchi University Graduate School of Medicine, Japan, 2 Department of Neurology, Tohoku University Graduate School of Medicine, 3 Department of Neurology, Tohoku Medical and Pharmaceutical University, 4 Department of Multiple Sclerosis Therapeutics, Fukushima Medical University
Backgournd: We previously reported the effect of sera from neuromyelitis optica （NMO）
patients on blood-brain barrier （BBB） dysfunction and the association between BBB dysfunction and glucose-regulated protein （GRP78） autoantibodies in NMO. Objective: To clarify the effect of IgG from anti-myelin oligodendrocyte glycoprotein antibodies （MOG-Abs）
associated disorders patients on the BBB-endothelial cell activation and the positivity of GRP78 in the disease. Methods: We purified IgG from sera with MOG-Abs associated disorder patients [15 sera in the acute phase （acute MOG）], 14 sera in stable stage （stable MOG）].
IgGs from 9 healthy and 27 disease controls were used as controls. IgG was exposed to the human brain microvascular endothelial cells （TY10） and the amount of nuclear NF-kB p65 positive cells as a marker of endothelial cell activation was analyzed using a high-content imaging systeme. Presence of GRP78 antibodies from patient IgGs was detected by western blots. Results: IgG in acute MOG group significantly induced the nuclear translocation of NF-kB p65 compared to those from stable MOG group and healthy/disease control group.
The rate of GRP78 antibody positivity observed in acute MOG groups （10/15, 66‰） was significantly higher than tha in the disease control groups （3/27, 11‰） or the healthy control groups （0/9, 0‰）. Conclusion: Endothelial cell activation induced by IgG incubtion was observed in not only NMO patients but also MOG associated disorders patients. GRP78 antibodies may be associated with BBB dysfunction in MOG-Abs associated disorders.
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MS-relatedTCRrecognizesCMVwhichprotects disabilityprogressioninpatientswithHLA-DRB1*04:05○FumieHayashi1,NorikoIsobe2,JacobGlanville3,
GuzailiayiMaimaitijiang1,TakuyaMatsushita1,Jun-ichiKira1
1 Department of Neurology, Graduate School of Medical Sciences, Kyushu University, Japan, 2 Department of Neurological Therapeutics, Graduate School of Medical Sciences, Kyushu University, 3 Computational and Systems Immunology Program, Stanford University School of Medicine
[Aim] To identify the multiple sclerosis （MS）-related antigens by analyzing T cell receptor （TCR） repertoire. [Methods] Peripheral blood mononuclear cells were obtained from 39 MS patients and 19 HCs. Next-generation sequencing （NGS） was conducted for complementary DNA for TCRβ chain. TCR diversity and TRBV/
J usage were analyzed. TCR motifs enriched in MS group compared to HCs were determined with a clustering method, GLIPH. We measured cytomegalovirus （CMV）
-IgG by ELISA and the percentage of CD4+CD25+ regulatory T cells （Treg） by flow cytometry. [Results] TCR diversity was decreased by age and was higher in MS （p = 0.039）. TRBV4-3 were more prevalent in MS. GLIPH consolidated 208,674 TCR clones of MS patients into 1,294 clusters. Out of the two clusters we identified, one was highly shared by HLA-DRB1*04:05 （+） MS patients （87.5%） and was predicted to recognize CMV （CMV-TCR）. The V usage of CMV-TCR was TRBV4-3. MS Severity Scores
（MSSS） were lower in the patients with CMV-TCR than in those without CMV-TCR （p
= 0.037）. In patients with HLA-DRB1*04:05, the CMV-IgG （+） group demonstrated lower MSSS than the CMV-IgG （-） group （p = 0.029）. CMV-infected patients tended to have higher proportion of Treg compared to the patients without CMV infection （p = 0.059）. [Conclusions] We characterized TCRs related to Japanese MS by applying NGS techniques and a TCR repertoire clustering tool. CMV is suggested to be related to HLA-DRB1*04:05 （+） MS, in a protective way partly via Treg. This is the first study in MS which refers to the association between CMV and HLA class II alleles.
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NeuronspromoteCD4+cellsinfiltrationinexperimentalautoimmuneencephalomyelitisviaCCL2
○YukiNakazato1,YukiFujita2,ToshihideYamashita2, MasamitsuNakazato1
1 Department of Internal Medicine, Division of Neurology, Respirology, Endocrinology, and Metabolism, Department of Internal Medicine, Faculty of Medicine, University of Miyazaki, Japan, 2 Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University
[Objective] An animal model of viral encephalitis revealed that neurons attract inflammatory cells via C-C motif chemokine ligand 2 （CCL2）. It remains unclear in multiple sclerosis （MS） whether neurons promote infiltration of inflammatory cells.
We have determined neuronal effect in murine model of experimental autoimmune encephalomyelitis （EAE） by modulating neuronal activity with genetic engineering methods. [Methods] We developed inhibitory designer receptors exclusively activated by designer drugs （DREADD） under a CaMKIIα promoter, and administered DREADD-carrying-AAV9 into the spinal cord. We applied targeted EAE, assessed daily EAE clinical scores for 28 days, and performed immunohistochemistry, in situ hybridization and quantitative PCR of cellular markers and inflammatory mediators in the spinal cord at peak of disease. We performed migration assay, and counted lymphocytes migrating toward neurons infected with AAV9. We knocked down Ccl2 mRNA with shRNA in targeted EAE mice and cortical neurons used in migration assay, and evaluated EAE score and histological analysis. [Results] Neuronal silencing mitigated EAE scores, reduced the expression of Ccl2 mRNA in the inflammatory lesions, and prevented migration of CD4+ cells toward neurons. Ccl2 shRNA administration to targeted EAE suppressed migration of CD4+ cells and alleviated the motor deficits of EAE. [Conclusions] Neuronal CCL2 produced by neuronal activation in EAE promotes migration of CD4+ cells and neuronal silencing by the inhibitory DREADD alleviates clinical scores and molecular markers of disease.
O-40-4
Connexin47regulatesautoimmunedemyelination inanovelmousemodelofmultiplesclerosis○RyoYamasaki,YinanZhao,SatoshiNagata,YukoNakamuta, HirooYamaguchi,Jun-ichiKira
Department of Neurology, Neurological Institute, Graduate School of Medical Sciences, Kyushu University, Japan
Objective: In multiple sclerosis plaques, oligodendroglial connexin （Cx） 47 constituting gap junction channels with astroglial Cx43 is persistently lost.
The roles of Cx47 remain undefined. We aimed to clarify the effects of Cx47 ablation on experimental autoimmune encephalomyelitis （EAE）. Methods:
We generated Plp1-CreERT;Cx47 fl/fl mice to produce tamoxifen-inducible oligodendroglia-specific conditional knockout of Cx47 （Cx47 icKO）. Cx47 icKO mice （n = 9） and their control littermates （Cx47 fl/fl） （n = 8） were immunized with myelin oligodendrocyte glycoprotein （MOG） peptide 35-55 to induce EAE.
Results: Cx47 icKO mice demonstrated exacerbation of acute and chronic EAE with increased relapse numbers. Cx47 ablation facilitated infiltration of Th17 cells into CNS lesions at acute phase, although MOG-specific proliferation of splenocytes was unaltered. Microarray analysis of isolated microglia revealed stronger activation with pro-inflammatory and injury-response phenotypes and enhanced expression of CCL2 in Cx47 icKO compared with Cx47 fl/fl mice at acute phase. Immunohistochemically, NOS2+ MHC class II+ microglia were more abundant in Cx47 icKO than Cx47 fl/fl mice. Cx47 icKO mice showed upregulation of A1-specific and pan-reactive genes, and more abundant astroglia harboring C3, a representative A1 marker, compared with Cx47 fl/
fl mice at acute phase. Conclusion: These findings suggest that Cx47 deletion augments acute CNS inflammation, causing relapsing and progressive EAE.