一般口演（若手道場）
若手道場 運動障害をきたす神経疾患
Wakate Dojo: Neural Disorders Causing Movement Disorders
座長：竹林 浩秀（新潟大学）・村松 里衣子（国立精神・神経医療研究センター） |
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| 2022年7月1日 10:00～10:15 沖縄コンベンションセンター 会議場B3・4 第6会場
2WD06m2-01
ドーパミンニューロン特異的BH4欠乏マウスの作製とジストニア発症機構の解析
Generation of dopaminergic neuron-selective BH4-deficient mice and analysis of the etiology of dopa-responsive dystonia
*小川 涼平(1)、鈴木 実乃里(1)、原 怜(1)、一瀬 宏(1)
1. 東京工業大学生命理工学院
*Ryohei Ogawa(1), Minori Suzuki(1), Satoshi Hara(1), Hiroshi Ichinose(1)
1. School of Life Science and Technology, Tokyo Institute of Technology, Tokyo, Japan
Keyword: dopa-responsive dystonia, Parkinson's disease, dopamine, tetrahydrobiopterin (BH4)
Dopamine (DA) is a neurotransmitter synthesized from tyrosine in the brain. Tyrosine hydroxylase (TH) catalyzes the rate-limiting step of DA synthesis. Tetrahydrobiopterin (BH4), which is an essential cofactor of TH, is synthesized from GTP through 3 enzymatic reactions. GTP cyclohydrolase I (GCH) is an enzyme catalyzes the first and rate-limiting step of BH4 de novo synthesis. Some mutations of GCH cause BH4 deficiency, inducing DA deficiency and dopa-responsive dystonia (DRD). However, it is unclear why symptoms of DRD are different from those of Parkinson’s disease (PD) even though both disorders are caused by DA deficiency in the brain.
To understand the etiology of DRD caused by BH4 deficiency, we have generated and analyzed some model mice with partial BH4 deficiency. However, it was difficult to analyze those mice in detail because they showed hyperphenylalaninemia and severe growth retardation due to systemic deficits of BH4 and monoamines. Hence, we generated DA neuron-specific Gch knock-out mice (Gch-cKO) by crossing DAT-Cre mice (expressing Cre recombinase in DA neurons) and Gch-flox mice (supplied from Dr. Keith Channon). Gch-cKO mice could survive after weaning and grew up into adults while their weight was approximately 10% less than control mice without Cre. The Gch expression was confirmed to be decreased in DA neurons by histochemical and biochemical analyses. BH4 and DA contents in the striatum and midbrain were obviously decreased compared to the control mice. In addition, behavioral tests revealed that Gch-cKO mice showed hypoactivity and bradykinesia.
The above results suggested that Gch-cKO mice can be utilized as dopaminergic neuron-selective BH4-deficient model mice. We are going to reveal the unsolved question of the etiology of DRD, such as sex difference in incidence of patients, diurnal fluctuation of symptoms, and the difference between dystonia and parkinsonism. | | 2022年7月1日 10:15～10:30 沖縄コンベンションセンター 会議場B3・4 第6会場
2WD06m2-02
ペランパネルによる神経活動の抑制がパーキンソン病モデルにおけるαシヌクレイン伝播を阻害する
Inhibition of neuronal activity with perampanel blocks α-synuclein transmission in Parkinson’s disease models
*上田 潤(1)、上村 紀仁(1)、髙橋 良輔(1)
1. 京都大学大学院医学研究科
*Jun Ueda(1), Norihito Uemura(1), Ryosuke Takahashi(1)
1. Kyoto University Graduate School of Medicine
Keyword: Parkinson’s disease, alpha-synuclein, neuronal activity, macropinocytosis
Objective: Parkinson’s disease is one of the most common neurodegenerative diseases. The pathological feature of Parkinson’s disease is progressive neuronal degeneration and the presence of Lewy bodies composed of misfolded α-synuclein. The intercellular transmission of pathogenic proteins plays a crucial role in Parkinson’s disease. Previous studies have demonstrated the correlation between neuronal activity and the neuronal uptake of pathogenic proteins in experimental models of neurodegenerative diseases. Therefore, we hypothesized that the inhibition of neuronal activity could modulate the dynamics of α-synuclein, inhibit the propagation of α-synuclein pathology, and attenuate the progression of Parkinson’s disease. Perampanel, an antiepileptic drug, is an AMPA receptor antagonist that inhibits neuronal activity. The objective of this study is to verify the efficacy of perampanel on α-synuclein transmission in Parkinson’s disease models. Methods: Mouse primary hippocampal neurons were transduced with α-synuclein preformed fibrils to examine the effect of perampanel, NBQX, and tetrodotoxin on the neuronal uptake of α-synuclein preformed fibrils. An in vivomodel of Parkinson’s disease was used to examine the effect of oral administration of perampanel on the neuronal uptake of α-synuclein preformed fibrils and the development of α-synuclein pathology. Results:Perampanel, NBQX, and tetrodotoxin blocked the neuronal uptake of α-synuclein preformed fibrils by inhibiting macropinocytosis. Furthermore, we showed that oral administration of perampanel inhibited the developmentof α-synuclein pathology in in vivo Parkinson’s disease model. Conclusion: Neuronal uptake of α-synucleinpreformed fibrils could be mediated by an activity-dependent mechanism, and targeting neuronal activity with perampanel could represent a new therapeutic strategy for Parkinson’s disease. | | 2022年7月1日 10:30～10:45 沖縄コンベンションセンター 会議場B3・4 第6会場
2WD06m2-03
遺伝性パーキンソン病原因分子LRRK2のI2020T変異がメンブレントラフィッキングに及ぼす影響
The influence of I2020T mutant LRRK2, the causal molecule of familial Parkinson's disease, on intracellular membrane trafficking
*服部 精人(1)、太田 悦朗(2,3,4,5)、永井 真貴子(6)、樋代 理子(1)、岩渕 和也(1)、岡野 栄之(5)
1. 北里大学大学院医療系研究科細胞免疫学、2. 北里大学医療衛生学部再生医療細胞デザイン研究施設細胞デザイン研究開発センター、3. 北里大学医療衛生学部免疫学Ⅱ、4. 北里大学大学院医療系研究科臨床免疫学、5. 慶應義塾大学医学部生理学、6. 北里大学医学部脳神経内科学
*Akito Hattori(1), Etsuro Ohta(2,3,4,5), Makiko Nagai(6), Riko Hidai(1), Kazuya Iwabuchi(1), Hideyuki Okano(5)
1. Program in Cellular Immunol, Gradu Sch Med Sci Kitasato Univ, 2. R&D center for Cell Design, Institute for Regenerative Medicine and Cell Design, Kitasato Univ, 3. Dept ImmunolⅡ, Kitasato Univ of Allied Health Sci, 4. Div Clinical Immunol, Gradu Sch Med Sci Kitasato Univ, 5. Dept Physiol Keio Univ Sch Med, 6. Dept Neurol, Kitasato Univ Sch Med
Keyword: Parkinson's disease, Membrane trafficking, iPS, Rab
パーキンソン病（PD）は中脳黒質のドーパミン産生神経細胞の脱落によって引き起こされる神経変性疾患である。北里大学では、日本の優性遺伝性PD 家系（S 家系）の発症原因がLeucine-Rich Repeat Kinase 2（LRRK2）のキナーゼドメイン内のI2020T 変異に起因することを発見し、LRRK2 の生理機能や基質分子を報告している。さらに、I2020T 変異LRRK2 をもつS 家系PD 患者から樹立したiPS 細胞（I2020T LRRK2-iPSC）由来神経細胞を用いて、ドーパミンの放出異常やリン酸化タウの増加などPD病態の一部をin vitro で再現可能であることを報告している。近年、メンブレントラフィッキング関連タンパク質であるRab群の機能異常が神経細胞死に関与することが注目されており、さらにLRRK2がこのRab群の一部を直接リン酸化し、機能調節することが報告されている。しかしながら、Rab群の機能異常に関する研究は、G2019S変異LRRK2やR1441C変異LRRK2をもつ遺伝性PD患者iPSCでは検証されているが、I2020T LRRK2-iPSCでは検証されていない。そこで我々は、I2020T 変異LRRK2とRab群がメンブレントラフィッキングに及ぼす影響を検証するために、ヒト神経芽細胞腫SH-SY5YおよびI2020T LRRK2-iPSCを用いてメンブレントラフィッキング解析法の検討を行った。その結果、蛍光ラベル標識トランスフェリン（555-Tf）を取り込ませたSH-SY5Yにおいて、細胞内の555-Tf およびRab群の局在解析ができる画像解析ソフトCellProfilerの解析パイプラインを構築した。さらに、この新規メンブレントラフィッキング解析法を用いて解析を進めた結果、I2020T変異LRRK2安定発現SH-SY5Yでは、SH-SY5Yおよび正常型LRRK2安定発現SH-SY5Yに比べ、Rab4、Rab5、Rab7のpuncta数、Intensityや細胞内局在に差異がある傾向を確認した。また、細胞に取り込ませた555-Tfのpuncta数やその細胞内局在に差異が確認された。次に、iPSC由来神経細胞を用いて同様の実験を行った結果、I2020T LRRK2-iPSC由来神経細胞では、ゲノム編集技術で修復したisogenic-iPSC由来神経細胞に比べ、Rab4、Rab7、Rab8、Rab10のpuncta数、Intensityや細胞内局在にそれぞれ差異がみられた。さらに、メンブレントラフィッキングの機能的な指標となるリン酸化Rab8、リン酸化Rab10においてもpuncta数、Intensityや細胞内局在にそれぞれ差異がみられた。555-Tfの細胞内挙動に関する実験は、現在解析中である。最後に、近年LRRK2が微小管を介したキネシンやダイニンなどモータータンパク質の機能調節に関与することが報告されているため、細胞内でのミトコンドリア動態を調べた。その結果、I2020T変異LRRK2安定発現SH-SY5Yでは、SH-SY5Yおよび正常型LRRK2安定発現SH-SY5Yに比べ、ミトコンドリアの細胞内移動距離に差異がある傾向を確認した。今後の研究展開として、iPSC由来神経細胞におけるマイトファジーを含めたミトコンドリアの解析、リン酸化Rab群に関する網羅的な解析を進めることによって疾患特異的な表現系を見出し、メンブレントラフィッキング異常を回復できうる治療候補薬剤の探索および評価系システムの構築に繋げていければと考えている。 | | 2022年7月1日 10:45～11:00 沖縄コンベンションセンター 会議場B3・4 第6会場
2WD06m2-04
筋萎縮性側索硬化症関連タンパク質VAPの細胞外機能と分泌メカニズム
Extracellular role and secretion mechanism of VAP, an ALS-related ER-resident protein
*亀村 興輔(1)、小園 梨央(1)、奥村 美紗子(1)、関根 清薫(2)、神山 大地(3)
1. 広島大学大学院統合生命科学研究科、2. 東北大学大学院生命科学研究科、3. ジョージア大学細胞生物学部
*Kosuke Kamemura(1), Rio Kozono(1), Misako Okumura(1), Sayaka Sekine(2), Daichi Kamiyama(3)
1. Grad Sch of Integr Sci for Life, Hiroshima Univ, 2. Grad Sch of Life Sci, Tohoku Univ, 3. Dept Cell Biol, Univ Georgia
Keyword: VAP, ALS, ER, Drosophila
VAP is a type II integral transmembrane protein localized at the endoplasmic reticulum (ER), and functions as a tethering protein of the membrane contact sites between ER and various intracellular organelles (e.g. Golgi apparatus, mitochondria). Recent studies revealed that VAP is cleaved, and its N-terminal MSP domain is secreted to the extracellular space. The secreted VAP MSP domain is known to bind to a variety of axon guidance receptors (Eph, Robo, Lar, etc.), implying that VAP could function non-cell autonomously. In addition, mutations in human VAPB were known to cause neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). However, as the VAP loss-of-function exhibits severe lethality in early developmental stages, the physiological functions of VAP, especially the extracellular functions of the VAP MSP domain are not well understood. In order to examine the roles of the secreted MSP domain of Vap33 (a Drosophila ortholog of VAP), we searched the amino acid sequences required for MSP secretion to generate the “unsecretable vap33 mutant” whose MSP is not secreted but can function intracellularly. We generated 25 Vap33 mutant constructs and found that MSP secretion from Vap33L118A-K122A mutant was significantly reduced. vap33-/- neurons show a morphological defect of the dendrite due to abnormal organelle distribution. Overexpression of Vap33L118A-K122A in the vap33-/- neurons rescued the dendrite defects, implying that the intracellular functions of Vap33L118A-K122A are likely to be normal. We generated vap33L118A-K122A mutant fly using CRISPR/Cas9 system, and found that vap33L118A-K122A mutant exhibited lethality at pharate adult. Furthermore, overexpression of wild type Vap33 in motor neurons rescued the lethality of vap33L118A-K122A mutant. From these results, we propose that Vap33 derived from motor neurons plays essential extracellular roles possibly to maintain muscle function required for emergence of pharate adult from pupa. Next, we conducted immunohistochemistry or biochemical experiments to explore the secretion mechanism of VAP. Using split-GFP system, we verified that Vap33 on the ER membrane directs its N-terminal MSP domain into cytosol. We further found that Vap33 inverts its topology at the plasma membrane. Therefore, VAP MSP domain is likely to be secreted through conventional secretion pathway accompanied by the topological conversion on the plasma membrane. | |
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