ALS/FTLD
O2-9-1-1 筋萎縮性側索硬化症におけるイノシトール6リン酸キナーゼ2の役割 Inositol hexakisphosphate kinase 2 is one of the candidate molecules as diagnostic marker for amyotrophic lateral sclerosis ○永田栄一郎1, 森谷祐介1, 藤井奈津子1, 小原さおり1, 佐藤忠之2, 高尾昌樹3, 潘雷4, 小川温子4, 秦野伸二4, 美原盤5, 瀧澤俊也1 ○Eiichiro Nagata1, Yusuke Moriya1, Natsuko Fujii1, Saori Kohara1, Tadayuki Satoh2, Masaki Takao3, Lei Pan4, Haruko Ogawa4, Shinji Hadano4, Ban Mihara5, Shunya Takizawa1 東海大学医学部内科学系神経内科1, 東海大学医学部教育・研究支援センター2, 東京都健康長寿医療センター神経病理学研究（高齢者ブレインバンク）3, 東海大学医学部基礎医学系分子生命科学4, 美原記念病院神経内科5 Dept Neurol, Tokai Univ Sch Med1, Edu and Res Suppt Cent, Tokai Univ Sch Med2, Dept Neuropath, Tokyo Metro Ins Geront3, Dept Mol Life Sci, Tokai Univ Sch Med4, Dept Neurol, Mihara Memorial Hosp5 Purpose: We previously reported that inositol hexakisphosphate kinase type 2 (InsP6K2), which converts inositol hexakisphosphate (InsP6) to Inositol pyrophosphates (InsP7), mediates cell death in neuronal cells. Hence, we investigate the role of InsP6K2 for mutant human superoxide dismutase transgenic mice (mSOD1 (G93A) Tg) and human spinal cords in amyotrophic lateral sclerosis (ALS) patients. Materials and methods: We used mSOD1 Tg at 12 weeks (before presenting motor neuron symptoms), and 17weeks (presenting motor neuron symptoms) and corresponding wild-type mice as same weeks. Spinal cord tissues of the mice were obtained and we did the quantitative real time PCR (qPCR) for InsP6K2 and the immunohistochemistory against anti-InsP6K2 antibody. Moreover, we also performed qPCR and the immunohistochemistory for InsP6K2 in fresh autopsy specimens from 6 sporadic ALS patients and 5 non-neurological patients (NNDP). Results: The expression of InsP6K2 in mSOD Tg at 17 weeks was 0.9 times as wild-type mice. However, the expression of InsP6K2 in mSOD1 Tg at 12 weeks mice was 1.3 times as wild-type mice. In the immunohistochemistry of InsP6K2, the nucleus was stained in wild-type mice, while the cytoplasm was stained in mSOD1 Tg. In the spinal cord of ALS patients, the expression level of InsP6K2 was lower compared to NNDP. Moreover, the number of anterior horn cells in spinal cord of ALS patients was significantly lower compared to NNDP. However, the ratio of the expression level of InsP6 K2 to an anterior cell was significantly higher compared to NNDP. Conclusions: These results suggest that InsP6K2 might be activated in ALS before presenting motor neuron disease symptoms and may play an important role of cell death mechanism on ALS. Moreover, InsP6K2 might be a potential biomarker of ALS.	O2-9-1-2 FUSはPSF/SFPQとの複合体形成を通じて、タウ遺伝子Maptのsplicingを制御しALS/FTLDの神経変性に関与する FUS regulates alternative splicing patterns of Mapt by cooperating with PSF/SFPQ in association with clinicopathological features of ALS/FTLD ○石垣診祐1, 藤岡祐介1, 宇田川剛1, 本田大祐1, 岡田洋平2, 勝野雅央1, 岡戸晴生3, 祖父江元1 ○Shinsuke Ishigaki1, Yusuke Fujioka1, Tsuyoshi Udagawa1, Daiyu Honda1, Yohei Okada2, Masahiro Katsuno1, Haruo Okado3, Gen Sobue1 名古屋大院・医・神経内科1, 慶應義塾大・医・生理学2, 都医学研・脳発達・神経再生3 Dept Neurol, Nagoya Univ, Nagoya1, Dept, Physiol, Keio Univ, Tokyo2, Tokyo Metropol Inst Med Sci, Tokyo3 FUS is linked to the pathogenesis of amyotrophic sclerosis (ALS), which is one of the most cruel neurodegenerative diseases. Mutations in Fus gene cause familial ALS and the protein mislocalization to the cytoplasm in the affected neurons are observed in both sporadic and familial ALS as well as FTLD which shares clinicopathological and genetical hallmarks with ALS. We found that FUS makes a high-molecular weight complex in the nucleus in the physiological condition of neurons and that is uncompleted in the ALS-associated mutants of FUS. The high-molecular complex of FUS consists of PFS/SFPQ that is a splicing factor in the nucleus. The interaction between FUS and PSF/SFPQ is affected in disease mutants. These observations suggest that compromised effects of FUS on alternative splicing could lead to neuronal cell degeneration in FUS-associated ALS/FTLD. From the exon-sensitive microarray analysis using Fus-silenced primary neurons, we identified Mapt exon10 is skipped by Fus. This alternative splicing event results in three-repeats (RD3) and four-repeats (RD4) Tau isoform production. We validated the results in both mouse primary neurons and human iPS-derived neurons to show no evolutional differences in mouse and human. Morphological abnormality of neurite was observed in Fus-silenced primary neurons and it was rescued by co-suppression of RD4. FUS-knock-down mice were established by injecting AAV encoding shRNA against Fus into bilateral hippocampus. The mice showed obvious reduction of FUS and increased ratio of RD4/RD3 in bilateral hippocampus. Abnormal anxiety behavior was observed in FUS-knock-down mice by behavior analysis including open filed test and elevated plus maze test. Pathological specimen analysis revealed that the change of the RD4/RD3 ratio b has been observed in FTLD and other tuopathies such as progressive supranuclear palsy (PSP). Thus, our findings suggest a clinicopatholigical and genetical link between FUS and Tau in ALS/FTLD.
O2-9-1-3 家族性筋萎縮性側索硬化症に見られる変異FUSタンパク質のアミロイド化促進メカニズム Amyloid-like fibrillation of FUS is triggered by a pathogenic mutation in a familial form of amyotrophic lateral sclerosis ○古川良明1, 野村尚生1, 渡辺祥司3, 金子貢巳2, 山中宏二3, 貫名信行2 ○Yoshiaki Furukawa1, Takao Nomura1, Shoji Watanabe3, Kumi Kaneko2, Koji Yamanaka3, Nobuyuki Nukina2 慶應・理工・生命機構化学1, 理研BSI・構造神経病理2, 理研BSI・運動ニューロン変性3 Dept Chem, Keio Univ, Yokohama, Japan1, Lab for Structural Neuropathology, RIKEN BSI, Wako, Japan2, Lab for Motor Neuron Disease, RIKEN BSI, Wako, Japan3 Dominant mutations in FUS (Fused in sarcoma) cause a familial form of amyotrophic lateral sclerosis (fALS), where abnormal accumulation of mutant FUS proteins in cytoplasm has been observed as a major pathological change. Many of pathogenic mutations have been shown to deteriorate the nuclear localization signal in FUS and thereby facilitate cytoplasmic mislocalization of mutant FUS proteins. Several other mutations, however, exhibit no effects on the nuclear localization of FUS in cultured cells, and their roles in the pathomechanism of fALS remain obscure. Here, we show that a pathogenic mutation, G156E, significantly increases the propensities for fibrillar aggregation of FUS. Recombinant human FUS was overexpressed in E.coli as a dual-tag protein with an N-terminal GST tag and a C-terminal polyhistidine tag, which facilitated the successful purification of a full-length FUS protein. Spontaneous formation of amyloid-like fibrillar aggregates was observed in G156E but not wild-type FUS, and notably, fibrils of mutant FUS was found to function as efficient seeds to trigger the aggregation of wild-type FUS. Also in cultured cells, wild-type FUS is diffusedly expressed in nucleus, while G156E mutation increases formation of nuclear foci. Based upon our results, therefore, we propose that FUS aggregation triggered by mutations play important pathological roles in a subset of FUS-related fALS cases.	O2-9-1-4 孤発性筋萎縮性側索硬化症におけるp62/SQSTM1遺伝子の解析 Analyses of the SQSTM1 gene encoding p62 in sporadic amyotrophic lateral sclerosis ○平野牧人1, 中村雄作2, 阪本光2, 上野周一2, 西郷和真3, 楠進3 ○Makito Hirano1, Yusaku Nakamura2, Hikaru Sakamoto2, Shuichi Ueno2, Kazumasa Saigoh3, Susumu Kusunoki3 近畿大学医学部堺病院・近畿大学医学部　神経内科1, 近畿大学医学部堺病院　神経内科2, 近畿大学医学部　神経内科3 Dept Neurology, Sakai Hospital Kinki Univ/Kinki Univ, Osaka1, Dept Neurology, Sakai Hospital Kinki Univ2, Dept Neurology, Kinki Univ3 Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with progressive muscle weakness and atrophy in limb, bulbar, respiratory regions. It occurs mostly sporadically, but 5%-10% of cases are familial. About ten causative genes in familial ALS have been identified. Mutations in such genes have been found occasionally in apparently sporadic cases. Recent reports have suggested that the SQSTM1 gene encoding p62 or sequestosome 1 is a causative gene for familial and sporadic ALS, accounting for 4.4% of all cases of sporadic ALS in the United States. Thus, we sequenced the promoter and coding regions of this gene in 70 Japanese patients with sporadic ALS. We found two novel missense mutations (p.Ala53Thr and p.Pro439Leu) in the coding region in two patients. The mutations were absent in 250 control subjects. Accumulating evidence demonstrates that p62 plays a role in protein degradation via proteasomes and autophagy, both of which are impaired in various neurodegenerative diseases. For example, this protein aggregates in neuronal and glial inclusions of Alzheimer disease, Parkinson disease, multiple system atrophy, and ALS. The presence of mutations in this gene found in the previous and preset reports demonstrates that p62 is not only a marker protein for aggregation, but also a key protein for neurodegeneration. Our finding that mutations are found in this racial population also suggests worldwide, common involvement of this gene in ALS.

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