一般口演（若手道場）
若手道場 治療法開発
Wakate Dojo: Therapeutic Development
座長：和氣 弘明（名古屋大学 院医・機能形態学）・篠崎 陽一（山梨大学） |
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| 2022年7月1日 14:00～14:15 沖縄コンベンションセンター 会議場B3・4 第6会場
2WD06a1-01
Rab9依存性新規オートファジー活性化によるiPS細胞由来VPS35 D620N変異ニューロン保護作用
Activation of Rab9-dependent alternative autophagy is neuroprotective in VPS35 D620N patient-derived iNeurons
*白石 朋敬(1,2)、坊野 恵子(1,2)、井口 保之(1)、岡野 ジェイムス洋尚(2)
1. 東京慈恵会医科大学内科学講座脳神経内科、2. 東京慈恵会医科大学再生医学研究部
*Tomotaka Shiraishi(1,2), Keiko Bono(1,2), Yasuyuki Iguchi(1), Hirotaka James Okano(2)
1. Department of Neurology, Jikei University School of Medicine, 2. Division of Regenerative Medicine, Jikei University School of Medicine
Keyword: PARKINSON'S DISEASE, AUTOPHAGY, VPS35
Background: VPS35 plays a key role in neurodegenerative processes in Alzheimer’s disease and Parkinson’s disease (PD). As many genetic studies have shown a close relationship between the autophagy and PD pathophysiology, PD-causal D620N mutation in VPS35 is known to impair autophagy. However, the molecular mechanisms underlying the neuronal cell death and autophagy impairment in PD are still under debate. Here, we found that Rab9-dependent “alternative” autophagy, which has a different molecular mechanism from ATG5/ATG7 dependent “conventional” autophagy, was impaired by the D620N mutation, and an activation of alternative autophagy decreased the vulnerability of VPS35 D620N patient-derived neuron. Methods: We established induced pluripotent stem cells (iPSCs) from the blood mononuclear cells of two PD patients with the D620N mutation in VPS35 and two healthy controls. All iPSC lines were differentiated into dopaminergic neurons (iNeurons) using only small molecules. Results: We first revealed that VPS35 D620N inhibits Rab9 localization to lysosomes using mCherry-EGFP-Rab9, an alternative autophagy reporter protein, in HeLa cells stably expressing VPS35 WT or D620N. Live cell imaging with VPS35-Venus and mCherry-Rab9 in HeLa cells revealed that the D620N mutation significantly decreased VPS35-Rab9 contact duration and fusion and fission frequency of Rab9 vesicles. In addition, autophagic vacuoles, which were assessed with Cyto-ID, significantly decreased in ATG5-knockdown VPS35 D620N iNeurons compared with ATG5-knockdown control iNeurons. Furthermore, estrogen, which is known to activate alternative autophagy, can improve Rab9 localization to lysosomes in the D620N HeLa cells and increased autophagic vacuoles in ATG5-knockdown VPS35 D620N iNeurons. Estrogen also increased the duration of VPS35-Rab9 contact and the fusion and fission frequency of Rab9 vesicles. Finally, we found that estrogen reduced the apoptosis in the VPS35 D620N iNeurons, and these neuroprotective effect of estrogen was diminished in Rab9 knockdown condition. Conclusion: To date, several epidemiological and experimental studies have reported the neuroprotective properties of estrogen on dopaminergic neurons in PD. Alternative autophagy might be important to maintain neuronal health and be associated with estrogen’s neuroprotective effect in PD with VPS35 D620N. | | 2022年7月1日 14:30～14:45 沖縄コンベンションセンター 会議場B3・4 第6会場
2WD06a1-03
セロトニン5-HT2A受容体刺激薬の抗うつ作用に関わる神経基盤研究
Neural bases underlying the antidepressant effect of serotonin 5-HT2A receptor agonists in mice
*高羽 里佳(1)、衣斐 大祐(1,2)、中齋 玄紀(1)、川瀬 りり菜(2)、北川 浩子(2)、吉田 圭佑(1,2)、間宮 隆吉(1,2)、北垣 伸治(1,2)、平松 正行(1,2)
1. 名城大学大学院薬学研究科、2. 名城大学大学薬学部
*Rika Takaba(1), Daisuke Ibi(1,2), Genki Nakasai(1), Ririna Kawase(2), Hiroko Kitagawa(2), Keisuke Yoshida(1,2), Takayoshi Mamiya(1,2), Shinnji Kitagaki(1,2), Masayuki Hiramatsu(1,2)
1. Grad. Sch. Fac of Pharm., Meijo Univ., 2. Dept. Sch. Fac of Pharm., Meijo Univ.
Keyword: serotonin 5-HT2A receptor, psilocin, lateral septum, treatment resistant depression
シロシビンは、大脳皮質の視覚野第Ⅴ層のセロトニン5-HT2A受容体（5-HT2AR）を刺激することで幻覚作用を誘発するが、最近、シロシビンが治療抵抗性うつ病患者に対して即効かつ持続的な治療効果を示すことが報告された。しかしながら、その神経基盤については不明である。そこで本研究では、シロシビンの5-HT2AR刺激作用に着目し、5-HT2AR刺激薬投与による抗うつ様作用およびそれに関連する責任脳部位や神経機構を調べた。 6～8週齢の雄性C57BL/6Jマウスに、シロシビンの活性代謝物であるシロシンを投与し、24時間後に抗うつ様作用を評価するため強制水泳試験（FST）を行ったところ、うつ様行動の指標として用いられる「無動時間」の有意な短縮が認められた。この作用は、ストレス応答や情動行動に関与する外側中隔核（LS）の5-HT2ARをノックダウンすることで消失し、一方、大脳皮質（視覚野第Ⅴ層）の5-HT2ARをノックダウンすると、シロシン投与による幻覚様行動（首振り運動）は抑制されたが、FSTにおける無動時間の短縮作用は影響を受けなかった。次に、LSにおける5-HT2AR発現神経細胞の詳細な解析のため、5-HT2ARプロモーター下にCreリコンビナーゼを発現するHtr2a-CreマウスのLSに、Cre依存的にGFPを発現させたところ、GFP陽性細胞は、GABA作動性神経マーカーと共局在した。さらに、LSのGFP陽性細胞から視床下部領域への投射があることを見出した。次にLSの5-HT2AR発現細胞の機能的役割を薬理遺伝学的に調べるためHtr2a-CreマウスのLSにCre依存的AAVによりhM3Dqを発現させ、人工リガンドであるclozapine N-oxide（CNO）投与によりLSの5-HT2AR発現細胞を活性化したところ、LSにおいてc-Fos発現が誘導され、さらにFSTにおける無動時間の短縮が認められた。 以上から、シロシビンなど5-HT2AR刺激薬は、LSの5-HT2ARを刺激し、GABA作動性神経を活性化させることで抗うつ作用を発揮すると推察された。現在、LSから視床下部領域に投射する5-HT2AR発現GABA作動性神経を光遺伝学的に操作させた際の行動学的および組織学的解析を進めている。本学会では、この結果もあわせて報告したい。 | | 2022年7月1日 14:45～15:00 沖縄コンベンションセンター 会議場B3・4 第6会場
2WD06a1-04
A novel structure of oligonucleotides administered intracerebroventricularly improves efficacy and alleviates acute toxicity in mice
*Su Su Lei Mon(1), Kotaro Yoshioka(1), Chunyan Jia(1), Taiki Kuineda(1), Yutaro Asami(1), Kie Yoshida-Tanaka(1), Wenying Piao(1), Hiroya Kuwahara(1), Kazutaka Nishina(1), Tetsuya Nagata(1), Takanori Yokota(1)
1. 1Department of Neurology and Neurological Science, Graduate School of Medical and Dental Sciences and Center for Brain Integration Research, Tokyo Medical and Dental University (TMDU), Tokyo, Japan
Keyword: Central nervous system, Antisense oligonucleotides, Intracerebroventricular injection, Overhanging duplex oligonucleotides
ASOs (antisense oligonucleotides) are short, synthetic, unmodified, or chemically modified nucleic acids that bind to the target gene and thereby alter the expression of the target RNA. Several types of ASOs, for example, steric-blocking ASOs such as Nusinersen, have been developed as novel therapeutics for intractable neurological diseases. However, gapmer-type ASOs have not been approved for clinical use due to insufficient knockdown potency and acute central nervous system toxicity. To overcome this hurdle, we developed a novel structure of double-stranded oligonucleotides, named as overhanging duplex oligonucleotides which are comprised of the original gapmer ASO and its extended complementary RNA strand. Here, we designed several ODOs targeting disease causing-genes such as Bace1 (beta-site amyloid precursor protein cleaving enzyme 1) and Mapt (microtubule-associated protein Tau) and analyzed its potency in the mouse brain in comparison to the original single-stranded ASOs via intracerebroventricular injection. It was noted that ODOs dose-dependently downregulated the target mRNAs more efficiently than the parent ASO in various areas of the mouse brain. Moreover, we clarified the optimal structure and chemical modifications of overhanging portion in ODO for the silencing potency in CNS. Above results have been published in FEBS Letters (Mon et al, January 2020). Furthermore, we evaluated acute CNS toxicity in mice injected with ASOs and ODOs using acute tolerability scoring system (ATSS) with PBS as the control group. ODOs were found to be able to significantly mitigate acute phenotypic toxicity in mice compared to ASO. In conclusion, this novel ODO technology provides a novel platform for the effective and safe treatment of neurological diseases. | | | |
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