一般ポスター
新技術と応用 |
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| 7月6日（木） 13:20-14:20 ポスター会場①
1P⑦-1
APEX2プロテオミクスを用いたFTLD/ALSにおけるC9orf72リピートRNA分解を促進するhnRNPA3近接分子の同定
APEX2 proteomics revealed a molecular which promotes hnRNPA3-mediated C9orf72 repeat RNA degradation in FTLD/ALS.
魚住 亮太1, 森 康治1, 後藤 志帆1, 宮本 哲愼1,2, 近藤 志都子1, 山下 智子1, 河邉 有哉1,3, 赤嶺 祥真1, 池田 学1
1. 大阪大学大学院・医学系研究科 精神医学教室, 2. 大阪府済生会茨木病院, 3. 箕面神経サナトリウム
Ryota Uozumi1, Kohji Mori1, Shiho Gotoh1, Tesshin Miyamoto1,2, Shizuko Kondo1, Tomoko Yamashita1, Yuya Kawabe1,3, Shoshin Akamine1, Manabu Ikeda1
1. Psychiatry, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan, 2. Saiseikai Ibaraki Hospital, Osaka, Japan, 3. Minoh Neuropsychiatric Hospital, Osaka, Japan
Aberrant expansion of GGGGCC repeat in the intron of the C9orf72 gene (C9) is the most frequent mutation in the spectrum pathology of familial FTLD and ALS, thereby called C9-FTLD/ALS. The transcribed C9 repeat RNA sequester RNA-binding protein (RBP)s into droplets formed by LLPS. So far, while many studies have supported that the repeat RNA is associated with C9-FTLD/ALS pathogenesis, the underlying mechanism of its degradation is still unclear.To investigate the pathway, here, we focused on hnRNPA3, one of the C9 repeat RBPs. hnRNPA3 do not appear to have RNA-degrading abilities, but an increased hnRNPA3 has reduced repeat RNA and its RAN translation products, DPR. Thus, hnRNPA3 might promote the degradation of repeat RNA. Furthermore, since hnRNPA3 undergo LLPS, we hypothesized that loose binding of hnRNPA3 with intracellular factors might facilitate hnRNPA3-mediated RNA degradation. To identify them, we introduced APEX2 proteomics that allowed selective biotinylation of proximate molecules. The biotinylated proteins were pulled down for subsequent LC-MS/MS. As a result of the following screenings, we identified one RBP and validated that its reduction led to the accumulation of repeat RNA, as quantified by qRT-PCR and in situ hybridization in both HeLa and C9 patient-derived cells.This study unveils the degradation pathway of the pathogenic repeat RNA in C9-FTLD/ALS. | | 7月6日（木） 13:20-14:20 ポスター会場①
1P⑦-2
マウス脳におけるタウ局所濃度決定方法の確立
Determination of local absolute concentration of tau in mouse brains
前田 希1, 堀 琴和1,2, 大江 洋平3, 宮坂 知宏1,2
1. 同志社大学大学院 生命医科学部 神経病理学研究室, 2. 同志社大学 神経変性疾患研究センター, 3. 同志社大学 生命医科学部 生体情報研究室
Nozomi Maeda1, Kotowa Hori1,2, Yohei Oe3, Tomohiro Miyasaka1,2
1. Neuropathology Laboratory, Graduate School of Life and Medical Sciences, Doshisha University, Kyoto, Japan, 2. Center for Research in Neurodegenerative diseases, 3. Biological information Laboratory, Graduate School of Life and Medical Sciences, Doshisha University
Tau is a microtubule-associated proteins that localized in the axons of neurons. In the brain of Alzheimer's disease (AD), tau is aggregated and deposited in cell body and dendrites of affected neurons. Therefore, in the pathogenesis of AD, change in local concentration of tau within neurons is occurred. Because of the pathology is reproduced only in the animal model with tau-over expression, there can be a threshold of tau concentration to form pathological inclusions. However, the absolute concentration in local regions in cells had not been able to be evaluated by technical limitation.We developed a agarose gel matrix which can bind the antigens, and form gel block. The gel block covalently bound with recombinant tau at known concentration was co-embedded in paraffin with brains from wild-type, and heterozygous and homozygous tau knockout mice. After immunostaining the sections with an anti-tau antibodies, the absolute amounts of tau concentration in tissues loci were quantified using standard curve obtained from the intensities of the gel blocks. The averaged tau concentrations in the mossy fibers were calculated to be 0.558 mg/cm3 in the wild type, 0.297mg/cm3 in the heterozygous, and 0.000 mg/cm3 in the homozygous tau knockout mice respectively.Here we demonstrated a novel procedure that can measure the local concentration of molecules within microscopic regions of cells. | | 7月6日（木） 13:20-14:20 ポスター会場①
1P⑦-3
簡易測定器を用いた神経細胞内K＋イオン濃度変化の測定
Measurement of intracellular K+ ion concentration changes in neurons using a simple measuring instrument
倉本 展行, 岡田 暉己, 岩本 昂也, 金城 俊彦, 宇野 恭介
摂南大学 薬学部 機能形態
Nobuyuki Kuramoto, Haruki Okada, Takaya Iwamoto, Toshihiko Kinjo, Kyosuke Uno
Lab. of Mol. Pharmacol. Setsunan Univ. Japan
The definition of intracellular ion concentration is possible using the patch clamp method, while the equipment is expensive. Fluorescence indicators for various ions are still in the development stage. In this study, we investigated whether it is possible to define intracellular and extracellular K+ concentrations and chase alterations in intracellular K+ concentrations using a simple K+ meter. A calibration curve of K+ concentration-ppm was prepared with a potassium chloride solution. We could determine the K+ concentration in tissue homogenates from several brain regions, and it was found that the K+ concentration per tissue weight was almost constant. It was found that when the cell suspension or the medium of adherent cells was changed to a medium containing no K+, the intracellular K+ concentration decreased. Also in the medium containing a high concentration of K+, the intracellular K+ concentration increased. The medium of the cell suspension and adherent cells was changed to a medium containing barium chloride, and intracellular K+ concentration was decreased in the barium chloride treatment. We will investigate using drugs that more directly change the intracellular K+ concentration (eg, K + channel opener). | | 7月6日（木） 13:20-14:20 ポスター会場①
1P⑦-4
アデノ随伴ウイルスベクターで使用できるコンパクトな抑制性ニューロン特異的プロモーターの探索
New inhibitory neuron-specific promoters for adeno-associated virus vectors
深井 悠貴1, 今野 歩1,2, 松崎 泰教1,2, 平井 宏和1,2
1. 群馬大学 医学系研究科 脳神経再生医学分野, 2. 群馬大学未来先端研究機構・ウイルスベクター開発研究センター
Yuuki Fukai1, Ayumu Konno1,2, Yasunori Matsuzaki1,2, Hirokazu Hirai1,2
1. Department of Neurophysiology and Neural Repair, Gunma University Graduate School of Medicine, 2. Viral Vector Core, Gunma University Initiative for Advanced Research (GIAR)
【Purpose】Recently, we identified a genomic region upstream of mouse GAD65 gene, which can be used as an inhibitory neuron-specific promoter (mGAD65 promoter). However, the mGAD65 promoter has 2.5 kb in length and occupies over a half of the accommodation space of adeno-associated virus (AAV) vector, which substantially compromises the usefulness of the mGAD65 promoter. Here, we developed compact inhibitory neuron-specific promoters available for AAV vectors.【Methods】We produced deletion constructs with different lengths from the 2.5-kb-mGAD65 promoter. AAV-PHP.eB, expressing GFP under the control of one of the shortened promoters, was intravenously administered to VGAT-tdTomato mice, which expressed tdTomato specifically in inhibitory GABAergic neurons. Three weeks after the viral injection, GFP expression in the brain was examined. Whether GFP was expressed in inhibitory neurons was verified by overlap of GFP fluorescence with that of tdTomato.【Result and Discussion】Some of the shortened promoters showed the same or higher promoter activity than that of the original mGAD65 promoter (2.5kb) without compromising the specificity to GABAergic neurons. The compact mGAD65 promoter allowed to express a larger transgene specifically in inhibitory neurons, and are of use for modulation of inhibitory neurons activity. | | 7月6日（木） 13:20-14:20 ポスター会場①
1P⑦-5
階層スフェロイド型ヒト血液脳関門モデルの薬物脳移行性評価試験への応用性検証
Applicability assessment of spheroidal human blood-brain barrier models for brain permeability assay of cyclic peptides
磯貝 隆斗1, 大木 聖矢1, 坂井 温斗1, 森尾 花恵1, 伊藤 慎悟2, 大槻 純男2, 降幡 知巳1
1. 東京薬科大学薬学部個別化薬物治療学教室, 2. 熊本大学大学院生命科学研究部微生物薬学分野
Ryuto Isogai1, Seiya Ohki1, Haruto Sakai1, Hanae Morio1, Shingo Ito2, Sumio Ohtsuki2, Tomomi Furihata1
1. Lab Clin Pharm & Exp Therapeut, Sch Pharm, Tokyo Univ Pharm & Life Sci., 2. Dpt Pharmaceu Microbiol, Faculty Life Sci, Kumamoto Univ.
In vitro human blood-brain barrier (BBB) models for BBB permeability evaluation of drugs are expected to play important roles in acceleration of drug development for CNS diseases. Accordingly, we have recently reported on development of a spheroidal BBB model based on human immortalized cells. In this study, we sought to clarify applicability of the model to drug BBB permeability studies through characterization of the kinetic properties of the SLS peptide, a novel BBB-permeable cyclic peptide.
After preparing the spheroidal BBB models, we first examined the temperature-dependency of the BBB uptake of FAM-labeled SLS peptides by measuring the fluorescence intensities inside the spheroids. The results showed that the BBB permeability levels at 60 min were higher at 37°C than those at 4°C, suggesting involvement of a putative carrier-mediated pathway in its BBB uptake. We also found that the carrier-mediated SLS peptide uptake showed a saturation trend in the time-dependency analyses (10-360 min), but did not in the concentration-dependency analyses (3-30 μM, 40 min).
In summary, using the spheroid BBB models, we have clarified the BBB permeability profile of the SLS peptides. Therefore, it can be expected that the spheroidal BBB models are useful for evaluation of the BBB permeability profile of cyclic peptides, presumably as well as other macromolecule drugs. | | | |
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