一般口演（Oral）

Oral ALS-2 一般口演 ALS-2

7月25日（木）10:00～10:15　第8会場（朱鷺メッセ　3F　303+304） 1O-08m2-1 筋萎縮性側索硬化症責任遺伝子産物TDP-43とFUSタンパク質はグアニン四重鎖との分子間相互作用で共通および異なる役割を持つ Akira Ishiguro（石黒亮）¹，Nobuyuki Kimura（木村展之）²，Akira Katayama（片山映）³，Takahide Kon（昆隆英）⁴ ¹法政大・マイクロナノテク ²国立長寿研・認知症先進医療・アルツハイマー・病因遺伝子 ³日医大・基礎医・生化・分生 ⁴大阪大院理生物科学・細胞構築 Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease caused by the degeneration of motor neurons leading to muscle weakness and atrophy. To date, more than 20 causative genes have been identified in hereditary ALS and some of them encode RNA-binding proteins, such as TDP-43 (TAR DNA-binding 43 kDa protein encoded by TARDBP) and FUS/TLS (fused in sarcoma/ translated in liposarcoma). Interestingly, both TDP-43 and FUS proteins have been identified as G-quadruplex binding protein. G-quadruplex structure is a non-canonical higher-order spatial structure formed by guanine rich nucleotide sequence that is built around tetrads of hydrogen-bonded guanine bases. The unique structure of RNA G-quadruplex acts as a signal of dendritic mRNAs for long distance transport and its local translation. Recent studies have revealed that G-quadruplex and the binding proteins are closely related to neurodegenerative disorder including ALS and frontotemporal lobar degeneration (FTLD). In order to identify the essential roles in the molecular mechanisms, we purified soluble form of TDP-43 and FUS proteins and analyzed the direct binding specificity. Although both of them interacted with common target G-quadruplex structure, showed different properties for binding specificity. TDP-43 has highly binding specificity and bind with only parallel-stranded RNA/DNA structure. However, FUS recognized not only parallel-stranded RNA/DNA, but also anti-parallel-stranded RNA/DNA and single stranded RNA/DNA. We confirmed the affect for the functions using a number of ALS-linked mutant proteins of TDP-43 and FUS in vitro and in primary neurons. We specified amino acid mutations that cause abnormalities in the interaction with G-quadruplex and mRNA transport. ALS-linked mutations may cause neurodegeneration due to functional toxicity or loss of function. These results suggest that TDP-43 and FUS have a common functional role but may contribute to distinct control depending on different RNA/DNA recognition mechanisms.		7月25日（木）10:15～10:30　第8会場（朱鷺メッセ　3F　303+304） 1O-08m2-2 家族性ALS/FTLD原因遺伝子によって引き起こされるTDP-43の凝集は2つの異なる経路によって生じる Seiji Watanabe（渡邊征爾），Kotaro Oiwa（大岩康太郎），Koji Yamanaka（山中宏二）名古屋大環境医病態神経 Mislocalization and cytosolic aggregation of TAR DNA-binding protein 43 (TDP-43) is a hallmark of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Although over 25 genes were identified as causative genes of inherited ALS/FTLD, the precise mechanism(s) to form TDP-43 inclusion bodies were not fully elucidated. In this study, we aimed to reveal intracellular pathway(s) involved in TDP-43 aggregation induced by the ALS/FTLD causative genes. Using a screening system with mouse neuroblastoma Neuro2a cells stably expressing GFP fused TDP-43 mutants lacking its nuclear localization signal (N2a·TDP(dNLS)-EGFP) and a mammalian expression library of the human ALS/FTLD causative genes, we classified the ALS/FTLD causative genes into three groups: 1. Genes whose products co-aggregate with TDP-43 (PFN1, FUS etc.), 2. Genes whose products aggregate without TDP-43 (VAPB, SQSTM1 etc.), 3. Genes whose products do not aggregate (TBK1, HNRNPA1 etc.). We also found that the group 1 genes were further divided into two sub-groups: 1) microtubule related proteins (MRPs) and 2) ribonucleotide binding proteins (RBPs). TDP-43 aggregation induced by MRPs was co-localized with aggresomal markers such as γ-tubulin and vimentin, and reduced by histone deacetylase 6 (HDAC6) suppression or nocodazole treatment. This suggests that MRPs inducing TDP-43 aggregates were formed in similar way of aggresomes. Whereas, RBPs induced TDP-43 aggregates were completely not affected by HDAC6 suppression or nocodazole treatment. Taken together, our findings suggest that TDP-43 aggregates, at least, in two distinct pathways; one is similar to aggresome formation and the other is not, in ALS/FTLD.

7月25日（木）10:30～10:45　第8会場（朱鷺メッセ　3F　303+304） 1O-08m2-3 TDP-43の光遺伝学的機能操作を用いたALS病態の再現 Kazuhide Asakawa（浅川和秀）^1,2，Koichi Kawakami（川上浩一）^1,2 ¹国立遺伝研遺伝形質発生遺伝 ²総研大院生命科学遺伝学 Cytoplasmic aggregation of the RNA-binding protein TDP-43 characterizes degenerating motor neurons in most cases of amyotrophic lateral sclerosis (ALS). However, exact mechanisms underlying TDP-43 toxicity that lead to neuronal dysfunction and degeneration remain elusive. Here, we provide evidence that enhancement of TDP-43 intermolecular interaction is sufficient to trigger its cytoplasmic mislocalization and destabilizes neuromuscular synapses in zebrafish. We develop a TDP-43 variant (optoTDP-43) harnessing a light-dependent oligomerization module of Cryptochrome 2 that increases its cytoplasmic pool and forms aggregates in light illumination- and cell type-dependent manners. Analyses of single spinal motor neurons reveal that a transient blue light illumination that causes cytoplasmic mislocalization, but not aggregation, of optoTDP-43 diminishes the subsequent motor axon outgrowth. Furthermore, live imaging of skeletal neuromuscular synapses demonstrates that the light illumination-dependent axon outgrowth defect is accompanied by an increase of collateral denervation frequency. These optogenetic interrogations point toward a causal link between TDP-43 oligomerization in the motor neurons and denervation of motor units, and provide a potential avenue for recapitulating pathologies of ALS and other TDP-43 proteinopathies in vivo in a spatiotemporally regulated fashion.		7月25日（木）10:45～11:00　第8会場（朱鷺メッセ　3F　303+304） 1O-08m2-4 FTLDスペクトラムにおけるSFPQの役割 Shinsuke Ishigaki（石垣診祐）^1,2，Yuichi Riku（陸雄一）^1,3，Yusuke Fujioka（藤岡祐介）¹，Minaka Ishibashi（石橋みなか）²，Satoshi Yokoi（横井聡）¹，Kuniyuki Endo（遠藤邦幸）¹，Nobuyuki Iwade（岩出展行）¹，Kaori Kawai（河合香里）^1,2，Hirohisa Watanabe（渡辺宏久）¹，Masahisa Katsuno（勝野雅央）¹，Mari Yoshida（吉田眞理）³，Gen Sobue（祖父江元）² ¹名古屋大院医神経内科 ²名古屋大院医神経変性認知症制御研究部 ³愛知医大加齢医科学研究所神経病理 Splicing factor, proline- and glutamine-rich (SFPQ) is an RNA-binding protein which functions for RNA metabolism including splicing and transcription. We have reported that Fused in sarcoma (FUS) and SFPQ regulate alternative splicing of Mapt gene at exon10 which generates 4-repeat tau (4R-tau)/ 3-repeat tau (3R-tau). Silencing of FUS or SFPQ resulted in abnormal FTLD-like behavioral impairments, accumulation of phosphorylated tau, and neuronal loss. The pathological investigation revealed that intra-nuclear co-localization of FUS and SFPQ was significantly reduced in the brain of ALS/FTLD and PSP compared to controls. On the other hand, no significant difference was found in AD cases. Exome sequencing in the database of JaCALS identified three independent single nucleotide variants in SFPQ in sporadic ALS patients. We also established an intranuclear proteome profile of SFPQ by immunoprecipitation analysis using pure nuclei extracted from neurons followed by LC/MS analysis. It shares several RNA-binding proteins with that of FUS including several familial ALS-associated molecules. Our findings suggest a pathophysiological link between SFPQ and the pathogenesis of a wide ranged spectrum of FTLD including ALS and PSP.