シンポジウム
02 優秀賞 受賞者企画シンポジウム
02 Symposium Organized by the Recipient of the 7th JSN Distinguished Investigator Award
座長：森 康治（大阪大学 大学院医学系研究科 精神医学） |
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| 2022年7月1日 14:00～14:24 沖縄コンベンションセンター 会議場B1 第3会場
2S03a-01
C9-ALS/FTDショウジョウバエモデルによるRAN翻訳制御機構の解明
Elucidation of regulatory mechanisms of RAN translation using C9-ALS/FTD fly model
*藤野 雄三(1,2)、上山 盛夫(1)、水野 敏樹(2)、永井 義隆(1)
1. 近畿大学医学部 脳神経内科、2. 京都府立医科大学大学院医学研究科 脳神経内科学
*Yuzo Fujino(1,2), Morio Ueyama(1), Toshiki Mizuno(2), Yoshitaka Nagai(1)
1. Department of Neurology, Kindai University Faculty of Medicine, 2. Department of Neurology, Kyoto Prefectural University of Medicine
Keyword: repeat-associated non-AUG translation, C9-ALS/FTD, RNA binding protein, Drosophila
The abnormal expansion of a GGGGCC repeat in intron of the C9orf72 gene causes familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) (C9-ALS/FTD). The expanded GGGGCC repeat RNAs transcribed from this mutated gene are translated into dipeptide repeat proteins (DPRs) by non-canonical repeat-associated non-AUG (RAN) translation. DPRs were detected in patients’ brain and some kinds of DPRs have been reported to be toxic, contributing to the pathogenesis of C9-ALS/FLD. However, the mechanisms that regulate RAN translation remain to be elucidated. To reveal the molecular mechanisms underlying RAN translation, we have established C9-ALS/FTD Drosophila model expressing the expanded GGGGCC repeat sequences and performed the following two genetic modifier screenings.
First, we checked the effect of overexpression of 18 candidates which had been reported to bind to GGGGCC repeat RNAs. As a result, we found RBP1 can alleviate eye degeneration and motor dysfunction of the C9-ALS/FTD Drosophila model. In biochemical assays, RBP1 decreased DPRs aggregates without altering the expression levels of GGGGCC repeat RNAs, suggesting its suppressive effect on RAN translation. These alleviating effects were abolished by mutations in the RNA-recognition motif of RBP1. On the contrary, knockdown of the Drosophila ortholog of RBP1 worsened eye degeneration and increased DPRs aggregates. Moreover, RBP1 directly bound to GGGGCC repeat RNAs and suppressed their translation in vitro. These results suggest that RBP1 suppresses RAN translation and repeat-induced toxicity through its binding ability to repeat RNAs.
Additionally, we performed another in vivo modifier screening for possible regulators of DPRs levels using candidate genes identified in recent genome-wide cellular screening. Among the 221 regulators, we selected 49 candidates that are reported to bind to GGGGCC repeat RNAs and/or to be involved in RNA metabolism. Two independent knockdown lines of each ortholog of these candidates were crossed with the C9-ALS/FTD Drosophila model, and the eye degeneration of offspring was examined. We found consistent modifying effects of both knockdown lines in 18 candidates. Some of candidates share functional pathways in RNA metabolism such as nucleocytoplasmic transport and RNA processing, suggesting their roles in regulating RAN translation. Our approach using Drosophila model will elucidate potential regulatory mechanisms of RAN translation. | | 2022年7月1日 14:24～14:48 沖縄コンベンションセンター 会議場B1 第3会場
2S03a-02
脊髄小脳失調症31型の原因となるUGGAAリピート標的低分子によるRNA毒性緩和
Alleviation of RNA toxicity by a small molecule targeting UGGAA repeat in spinocerebellar ataxia type 31
*柴田 知範(1)、長野 来南(2)、上山 盛夫(3)、二宮 賢介(4)、廣瀬 哲郎(4)、永井 義隆(3)、石川 欽也(5)、河合 剛太(2)、中谷 和彦(1)
1. 大阪大学産業科学研究所、2. 千葉工業大学工学研究科、3. 近畿大学医学研究科、4. 大阪大学生命機能研究科、5. 東京医科歯科大学
*Tomonori Shibata(1), Konami Nagano(2), Morio Ueyama(3), Kensuke Ninomiya(4), Tetsuro Hirose(4), Yoshitaka Nagai(3), Kinya Ishikawa(5), Gota Kawai(2), Kazuhiko Nakatani(1)
1. SANKEN, Osaka Univ, 2. Fac of Eng, Chiba Inst of Tech, 3. Sch of Med, KINDAI Univ, 4. FBS, Osaka Univ, 5. Tokyo Med & Dent Univ
Keyword: Spinocerebellar ataxia type 31, RNA toxicity, Repeat RNA, RNA-targeting small molecule
Spinocerebellar ataxia type 31 (SCA31) is an autosomal dominant spinocerebellar degeneration caused by aberrant insertion of TGGAA repeat into intronic region shared by BEAN1and TK2genes. Transcription of the inserted (TGGAA)nproduced toxic r(UGGAA)n, resulting in accumulation of RNA foci accompanied by sequestration of RNA-binding proteins (RBPs). In patients with SCA31, RNA foci containing UGGAA repeat are found in the nucleus of Purkinje cells. Furthermore, UGGAA repeat-mediated RNA toxicity has been demonstrated by phenotypic assay using Drosophilamodel of SCA31. Therefore, the pathogenic mechanism of SCA31 is thought to be gain-of-function of UGGAA repeat. Precedent study demonstrated that overexpression of TDP-43, which is found as UGGAA repeat-binding protein, disrupt the accumulation of RNA foci and improved disease phenotype of Drosophilamodel of SCA31. The alleviation of UGGAA repeat-mediated RNA toxicity by UGGAA repeat-binding proteins suggests the potential for feasible therapeutic approach for SCA31 by targeting toxic UGGAA repeat with small molecules. In order to explore UGGAA repeat-binding small molecules, we have screened our in-house chemical library containing nucleic acid-binding small molecules by surface plasmon resonance assay and found naphthyridine carbamate dimer (NCD). The NMR structural analysis of NCD-bound RNA complex clarified that two NCD molecules bound to 5’-GGA-3’/3’-AGG-5’ internal loop in 5’-UGGAA-3’/3’-AAGGU-5’ pentad by the formation of hydrogen bonding pairs of guanines with naphthyridines. We next examined the inhibitory effect on the interaction of UGGAA repeat with RBPs and the formation of RNA foci by NCD, demonstrating that NCD interfered with the RNA–RBP interactions and the accumulation of RNA foci in nucleus. Finally, we examined the effect of NCD on phenotype in Drosophilamodel of SCA31 which exhibit compound eye degeneration induced by expression of UGGAA repeat. Feeding NCD to larvae of Drosophilamodel of SCA31significantly alleviated degeneration of compound eyes in adults. These studies demonstrate that small molecules targeting toxic repeat RNAs have a therapeutic potential for repeat expansion diseases. | | 2022年7月1日 14:48～15:12 沖縄コンベンションセンター 会議場B1 第3会場
2S03a-03
C9orf72-FTD/ALSにおけるDNA損傷修復障害
DNA damage repair impairment in C9orf72-FTD/ALS
*二瓶 義廣(1)
1. 慶應義塾大学医学部
*Yoshihiro Nihei(1)
1. Keio University School of Medicine
Keyword: C9orf72, DNA damage, dipeptide-repeat proteins, phosphorylated Ataxia Telangiectasia Mutated
DNA damage accumulation is a wide spread phenomenon in aged brains and it is more prevalent in the brains of neurodegenerative diseases. There are several types of DNA damage: DNA double-strand breaks (DSBs) are the most severe type of DNA damage and often lead to cell death if not repaired correctly. In mammalian cells, homologous recombination (HR) and non-homologous end joining (NHEJ) are the two major cellular mechanisms that repair DSBs. HR fully restores the original sequence using the sister chromatid as a template, but is restricted to the late S- to G2/M-phase of the cell cycle. NHEJ is a rather error-prone repair mechanism that recombines truncated DNA ends without template DNA, and thus can occur throughout the cell cycle. Since neurons are arrested in G0-phase and are non-proliferative, their main repair mechanism is limited to NHEJ. With such error-prone repair pathways, neurons are highly vulnerable to DNA damage.
Repeat expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Expanded sense and antisense repeat RNA transcripts in C9orf72 are translated into five kinds of dipeptide-repeat proteins (DPRs) in an AUG-independent manner. Recent studies have revealed expanded-repeat RNA transcripts and DPRs induce DNA damage. Expanded C9orf72 repeat RNA transcripts form G-quadruplex structures and promote the formation of RNA:DNA hybrids (R-loops), which are prone to DSBs. Among five DPRs, three DPRs (poly-glycine–alanine: poly-GA, poly-glycine–arginine: poly-GR, poly-proline-arginine: poly-PR) are known to be toxic and induce DNA damage repair impairment. In an in vitro model, these three DPRs commonly increase DSBs. Poly-GR and poly-PR increase foci formed by phosphorylated Ataxia Telangiectasia Mutated (pATM), a major sensor of DSBs, whereas poly-GA evokes a reduction of pATM foci. In dentate gyri of C9orf72 patients, enhanced poly-GA deposition correlates with reduced pATM foci. Since cytoplasmic pATM deposits partially colocalize with poly-GA deposits, these results suggest that poly-GA, the most frequent DPR observed in C9orf72 patients, differentially causes DNA damage and that poly-GA selectively sequesters pATM in the cytoplasm inhibiting its recruitment to sites of DNA damage. In summary, the C9orf72 repeat expansion products exacerbate DNA damage in a variety of ways, causing neurodegeneration. | | 2022年7月1日 15:12～15:36 沖縄コンベンションセンター 会議場B1 第3会場
2S03a-04
C9orf72遺伝子由来poly(PR)ペプチドの構造機能連関
Order controls disordered droplets: structure-function relationships in C9ORF72-derived poly(PR)
*金蔵 孝介(1)、早水 裕平(2)、黒田 雅彦(1)
1. 東京医科大学、2. 東京工業大学
*Kohsuke Kanekura(1), Yuhei Hayamizu(2), Masahiko Kuroda(1)
1. Tokyo Medical University, 2. Tokyo Insitute of Technology
Keyword: Amyotrophic Lateral Sclerosis, C9orf72, dipeptide repeat protein, liquid liquid phase separation
Dipeptide repeat proteins (DRPs) are produced from the mutant C9ORF72, the most prevalent causative gene of ALS, via RAN translation. Among them, R-rich DRPs tend to undergo phase-separation due to the repetitive structure of positively charged R and are thought to cause ALS by interfering with the function of membraneless organelles. However, not all R-rich peptides are toxic, and it was unclear why poly(PR), which has a simple structure, acquires toxicity. We investigated the structure-function relationship of R-rich DRPs to understand the molecular mechanism underlying it. First, we generated (PR)12 mutants with altered charge distribution without changing the net charge and tested the inhibitory effect of the mutants on protein translation. (PR)12 effectively suppressed protein translation whereas P12R12 totally lost the inhibitory effect. We performed a quantitative proteomics to elucidate the molecular mechanism and showed that while the proteomes of (PR)12 and R12 were qualitatively unchanged, quantitative changes occurred up to thousands of times, and the effect was partially lost in the P12R12. Furthermore, the proteome data showed that the proteins enriched in (PR)12 proteomics contained a large number of acidic amino acids, and these proteins underwent phase-separation with (PR)12, but not with R12 or P12R12. In addition, the fluidity in the phase-separated droplets formed by (PR)12 was much higher than that of R12 and P12R12 droplets. We hypothesize that the insertion of Pro into the consecutive Rs inhibits the strong intermolecular interaction and weakens the binding energy but instead promotes multivalent binding, which allows phase separation with various proteins and interfere with the functions, resulting in toxicity. We next investigated why the degree of toxicity and the toxicity mechanism is partially different between poly(PR) and poly(GR). G has a small side chain and does not inhibit the degree of freedom of R, whereas P significantly inhibits the degree of freedom of R. Therefore, we believe that poly(GR) has relatively similar biological properties to poly(R), whereas poly(PR) has different properties. In this symposium, we would like to introduce the structure-function relationship of R-rich DRPs that we have been working on. | | 2022年7月1日 15:36～16:00 沖縄コンベンションセンター 会議場B1 第3会場
2S03a-05
神経疾患におけるグアニン四重鎖の細胞内機能
The cellular functions of G-quadruplex in neurological diseases
*塩田 倫史(1)
1. 熊本大学発生医学研究所
*Norifumi Shioda(1)
1. Kumamoto University, IMEG, Japan
Keyword: G-quadruplex, neurological disorders, nucleic acid secondary structures
G-quadruplex (G4) is a unique nucleic acid structure that formed when a four-stranded structure is produced within a single-stranded guanine-rich sequence. Four guanine molecules form a square planar arrangement, termed G-quartet, which are stacked on top of each other to form the G4 structure in DNA (G4DNA) and in RNA (G4RNA). Recent studies have revealed that G4DNA and G4RNA are folded in cells, which suggested their biological and pharmacological significance in DNA replication, transcription, epigenetic modification, and RNA metabolism. So far, we have reported the following; 1) G4 is a target of cognitive function therapy for ATR-X intellectual disability syndrome, in which mutations are found in a G4 binding protein ATRX. 2) G4 is formed in heterochromatin depending on neuronal development. 3) G4 promotes prionoids in a CGG triplet repeat disease, Fragile X-associated tremor/ataxia syndrome (FXTAS). 4) 5-aminolevulinic acid is a potential candidate drug for treating some neurological diseases through the G4 binding ability. In this symposium, I will summarize the significant roles of G4 in neurological diseases. | | | |
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