一般口演

一般口演5

2021/9/30　17:00～18:00　オンデマンド D会場 O5-1 ドパミンD2受容体と脂肪酸結合タンパク質FABP3に依存する α シヌクレインの新規伝播機構 Impact of dopamine D2 receptor through fatty acid-binding protein 3 in α-synuclein propagation ^○川畑伊知郎, Kohji Fukunaga 東北大学大学院薬学研究科先進脳創薬講座 ^○Ichiro Kawahata, Kohji Fukunaga Department of CNS drug innovation, Tohoku University α-Synuclein accumulation into dopaminergic neurons is a pathological hallmark of Parkinson's disease. Fatty acids partially regulate α-synuclein accumulation, and fatty acid-binding protein 3 (FABP3) associates with the aggregation. FABP3 is rich in dopaminergic neurons and interacts with dopamine D2 receptors, specifically the long type (D2L), abundant in caveolae. Here, we investigated the impact of dopamine D2L receptors and FABP3 in the uptake process of α-synuclein. We employed mesencephalic neurons derived from dopamine D2L^-/-, dopamine D2 receptor null (D2 null), FABP3^-/-, and wild-type C57BL6 mice, and analyzed the uptake ability of fluorescence-conjugated α-synuclein monomers and fibrils. We found that D2L receptors are co-localized with FABP3. Immunocytochemistry revealed that tyrosine hydroxylase (TH)⁺ FABP3^-/-, D2L^-/- or D2 null neurons do not take up α-synuclein monomers. The deletion of α-synuclein C-terminus completely abolished the uptake to dopamine neurons. Likewise, dynasore, a dynamin inhibitor, and caveolin-1 knockdown also abolished the uptake. Additionally, D2L and FABP3 were critical for α-synuclein fibrils uptake. D2L and accumulated α-synuclein fibrils were well co-localized. Aggregated fibrils accelerated TH proteasomal degradation. Intriguingly, FABP3 was essential for MPP⁺-induced reduction of mitochondrial activity and the production of reactive oxygen species. These data indicate that dopamine D2L with a caveola structure coupled with FABP3 is critical for α-synuclein uptake by dopaminergic neurons and the loss of neuronal homeostasis, suggesting a novel pathogenic mechanism of synucleinopathies, including Parkinson's disease.		2021/9/30　17:00～18:00　オンデマンド D会場 O5-2 αシヌクレイン単量体の構造状態が、フィブリル構造多型と疾患多型を規定する The conformational state of α-synuclein monomer defines fibril structure polymorphisms and disease ^○AGUIRRE CESAR, 池中健介大阪大学大学院医学系研究科 ^○CESAR AGUIRRE, 健介池中 Department of Neurology, Graduate School of Medicine, Osaka University The aggregation of ɑ-synuclein (aSyn) is a pathological hallmark of several neurodegenerative disorders, including Parkinson’s disease (PD), Lewy body disease, and Multiple system atrophy (MSA). Although these disorders are caused by the accumulation of the same protein, their clinical and pathological characteristics are completely different. Recent studies suggest that variations in the fibril structure of aSyn fibril may associated with the variations in disease phenotypes. However, the molecular mechanisms of aggregation that lead to the formation of specific fibril morphologies remain elusive. In this study, we unexpectedly found that aSyn can create different types of fibril even from a same buffer in physiological condition. Using a combination of ThT assay and TEM, we found two classes of WT-αSyn fibrils with distinctive kinetics and morphologies, namely Fibrils A and B. An analysis of monomeric solutions of aSyn by SEC, AUC, SAXS and NMR showed that this protein can exist in two molecular populations in equilibrium that lead to distinct morphologies in the fibrillation process. Currently, we are investigating how the molecular equilibrium is involved in the development of distinctive synucleinopathies, such as PD and MSA.

2021/9/30　17:00～18:00　オンデマンド D会場 O5-3 パーキンソン病の臨床バイオマーカーおよび薬剤スクリーニングへのα-シヌクレイン凝集体増幅法の応用 Application of the amplification assay of α-synuclein aggregates to clinical biomarker and drug screening for Parkinson's disease ^○角田渓太, Kensuke Ikenaka, Makoto Hideshima, Yasuyoshi Kimura, César Aguirre, Hideki Mochizuki 大阪大学大学院医学系研究科　神経内科学 ^○Keita Kakuda, Kensuke Ikenaka, Makoto Hideshima, Yasuyoshi Kimura, César Aguirre, Hideki Mochizuki Department of Neurology, Osaka University The accumulation of misfoldedα-synuclein(aSyn) aggregates have been considered as a main pathogenesis of Parkinson’s disease(PD). Detection of aSyn aggregates from biological fluid can be a clinical biomarker, and inhibition of aggregating process can be the target for developing of the disease modifying therapy. Here, we report the protein misfolding amplification assay (PMCA) detecting aSyn aggregates from cerebrospinal fluid (CSF) of the patients by HANdai Amyloid Burst Inducer (HANABI), and two-step screening of PMCA and cell-based assay to identify the inhibitor of aggregation. First, we confirmed that HANABI assay can detect minute amounts of aSyn aggregates as seeding activity in dose-dependent manner. The seeding activity of CSF from patients with PD was higher than that of controls, and significantly correlated with the MIBG heart-to-mediastinum ratio, which reflects Lewy pathology in peripheral nerves. These findings reconfirmed that the aggregation of aSyn play a key role in pathogenesis of PD. Nest, we performed two-step screening seeking for the compounds that have inhibitory effects on aSyn aggregation from 1,262 FDA-approved small compounds library. The first screening based on PMCA identified 30 hit-compounds, and the cell-based second screening found seven compounds that prevent aSyn aggregation without substantial cell-toxicity. Among final seven compounds, we focused on tannic acid as the most promising one, and the effect was also validated by Caenorhabditis elegans model. In conclusion, the amplification assay of aSyn aggregates can be applied to the measurement of the degree of aSyn aggregation of patients, and also to the drug screening that inhibits aggregation and thus prevents the disease progression of PD.		2021/9/30　17:00～18:00　オンデマンド D会場 O5-4 RNAグアニン四重鎖はα-シヌクレイン相転移を誘導する RNA G-quadruplexes induce phase transition of α-synuclein ^○矢吹悌¹, Kazuya Matsuo¹, Kohei Maeda¹, Tomohiro Mizobata¹, Yasushi Kawata¹, Sefan Asamitsu¹, Norifumi Shioda¹ 1.熊本大学発生医学研究所ゲノム神経学分野, 2.Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University ^○Yasushi Yabuki¹, Kazuya Matsuo¹, Kohei Maeda¹, Tomohiro Mizobata¹, Yasushi Kawata¹, Sefan Asamitsu¹, Norifumi Shioda¹ 1.Department of Genomic Neurology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, 2.Department of Biomedical Science, Institute of Regenerative Medicine and Biofunction, Graduate School of Medical Science, Tottori University Aggregation of α-synuclein (α-Syn) triggers development of α-synucleinopathies such as Parkinson’s disease and Dementia with Lewy Bodies. It has known that α-Syn can propagate in the brain, like as prion protein. However, the molecular mechanisms remain unclear. Here, we show that RNA G-quadruplexes (G4RNAs) facilitate α-Syn aggregation through the liquid-to-solid transition. Electrophoretic mobility shift assay revealed that α-Syn significantly and preferentially formed a complex with G4RNAs than with other RNAs (mutated G4RNAs, CAG hairpin form and polyA). Purified α-Syn formed liquid droplets by LLPS, and liquid-to-solid transition of α-Syn significantly facilitated by adding G4RNAs under the condition of molecular crowding (15% polyethylene glycol). Moreover, cellular stress markedly increased G4RNA foci and in turn caused α-Syn aggregates in α-Syn-expressed cells. These observations suggest that α-Syn may undergoes phase transition by G4RNAs under the pathological condition in vivo. We will investigate the mechanism underlying phase transition of α-Syn in cellulo and in vivo.