2022/01/27 更新

写真a

ヤマナカ トモユキ
山中 智行
YAMANAKA Tomoyuki
所属
脳研究所 生命科学リソース研究センター 准教授
職名
准教授
外部リンク

学位

  • 博士(医学) ( 2002年3月   横浜市立大学 )

研究キーワード

  • 神経疾患

  • NF-Y

  • 転写因子

  • HSP70

  • ハンチントン病

  • 凝集体

  • ポリグルタミン

  • ハンチンチン

  • 神経変性

研究分野

  • ライフサイエンス / 神経科学一般

  • ライフサイエンス / 神経形態学

  • ライフサイエンス / 神経科学一般

経歴(researchmap)

  • 新潟大学   脳研究所附属生命科学リソース研究センター   准教授

    2021年5月 - 現在

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  • 同志社大学脳科学研究科   認知記憶加齢部門   准教授

    2015年4月 - 2021年4月

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  • 順天堂大学   神経変性疾患病態治療探索講座   協力研究員(兼任)

    2013年4月 - 2015年3月

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  • 独立行政法人理化学研究所 構造神経病理研究チーム   研究員

    2010年4月 - 2015年3月

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  • 独立行政法人理化学研究所 構造神経病理研究チーム   基礎科学特別研究員

    2007年4月 - 2010年3月

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  • 独立行政法人理化学研究所 構造神経病理研究チーム   研究員

    2005年3月 - 2007年3月

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  • 横浜市立大学医学部 分子細胞生物学教室   博士研究員

    2003年4月 - 2005年2月

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  • 横浜市立大学医学部 分子細胞生物学教室   学術振興会特別研究員(PD)

    2002年4月 - 2003年3月

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  • 横浜市立大学 医学研究科 修了 博士(医学)の学位取得

    2002年3月

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  • 東京理科大学 薬学研究科 修了   Graduate School of Pharmaceutical Sciences

    1999年3月

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  • 東京理科大学 薬学部 薬学科 卒業   Faculty of Pharmaceutical Sciences, Department of Pharmacy

    1997年3月

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▶ 全件表示

経歴

  • 新潟大学   脳研究所 生命科学リソース研究センター   准教授

    2021年5月 - 現在

 

論文

  • Mutant VAPB: Culprit or Innocent Bystander of Amyotrophic Lateral Sclerosis? 招待 査読

    Nica Borgese, Francesca Navone, Nobuyuki Nukina, Tomoyuki Yamanaka

    Contact   4   251525642110225 - 251525642110225   2021年1月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:SAGE Publications  

    Nearly twenty years ago a mutation in the VAPB gene, resulting in a proline to serine substitution (p.P56S), was identified as the cause of a rare, slowly progressing, familial form of the motor neuron degenerative disease Amyotrophic Lateral Sclerosis (ALS). Since then, progress in unravelling the mechanistic basis of this mutation has proceeded in parallel with research on the VAP proteins and on their role in establishing membrane contact sites between the ER and other organelles. Analysis of the literature on cellular and animal models reviewed here supports the conclusion that P56S-VAPB, which is aggregation-prone, non-functional and unstable, is expressed at levels that are insufficient to support toxic gain-of-function or dominant negative effects within motor neurons. Instead, insufficient levels of the product of the single wild-type allele appear to be required for pathological effects, and may be the main driver of the disease. In light of the multiple interactions of the VAP proteins, we address the consequences of specific VAPB depletion and highlight various affected processes that could contribute to motor neuron degeneration. In the future, distinction of specific roles of each of the two VAP paralogues should help to further elucidate the basis of p.P56S familial ALS, as well as of other more common forms of the disease.

    DOI: 10.1177/25152564211022515

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    その他リンク: http://journals.sagepub.com/doi/full-xml/10.1177/25152564211022515

  • Gene expression profiling in neuronal cells identifies a different type of transcriptome modulated by NF-Y. 査読 国際誌

    Tomoyuki Yamanaka, Haruko Miyazaki, Asako Tosaki, Sankar N Maity, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    Scientific reports   10 ( 1 )   21714 - 21714   2020年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    A heterotrimeric transcription factor NF-Y is crucial for cell-cycle progression in various types of cells. In contrast, studies using NF-YA knockout mice have unveiled its essential role in endoplasmic reticulum (ER) homeostasis in neuronal cells. However, whether NF-Y modulates a different transcriptome to mediate distinct cellular functions remains obscure. Here, we knocked down NF-Y in two types of neuronal cells, neuro2a neuroblastoma cells and mouse brain striatal cells, and performed gene expression profiling. We found that down-regulated genes preferentially contained NF-Y-binding motifs in their proximal promoters, and notably enriched genes related to ER functions rather than those for cell cycle. This contrasts with the profiling data of HeLa and embryonic stem cells in which distinct down-regulation of cell cycle-related genes was observed. Clustering analysis further identified several functional clusters where populations of the down-regulated genes were highly distinct. Further analyses using chromatin immunoprecipitation and RNA-seq data revealed that the transcriptomic difference was not correlated with DNA binding of NF-Y but with splicing of NF-YA. These data suggest that neuronal cells have a different type of transcriptome in which ER-related genes are dominantly modulated by NF-Y, and imply that NF-YA splicing alteration could be involved in this cell type-specific gene modulation.

    DOI: 10.1038/s41598-020-78682-8

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  • Proteomic Analysis of Subcellular Compartments Containing Disseminated Alpha-synuclein Seeds 査読

    Junya Kasahara, Yukio Imamura, Akiko Hiyama, Tomoyuki Yamanaka, Haruko Miyazaki, Nobuyuki Nukina

    Neuroscience Research   in press   2020年12月

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  • FACS-array-based cell purification yields a specific transcriptome of striatal medium spiny neurons in a murine Huntington disease model. 査読 国際誌

    Haruko Miyazaki, Tomoyuki Yamanaka, Fumitaka Oyama, Yoshihiro Kino, Masaru Kurosawa, Mizuki Yamada-Kurosawa, Risa Yamano, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    The Journal of biological chemistry   295 ( 29 )   9768 - 9785   2020年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin gene. Results from previous studies have suggested that transcriptional dysregulation is one of the key mechanisms underlying striatal medium spiny neuron (MSN) degeneration in HD. However, some of the critical genes involved in HD etiology or pathology could be masked in a common expression profiling assay because of contamination with non-MSN cells. To gain insight into the MSN-specific gene expression changes in presymptomatic R6/2 mice, a common HD mouse model, here we used a transgenic fluorescent protein marker of MSNs for purification via FACS before profiling gene expression with gene microarrays and compared the results of this "FACS-array" with those obtained with homogenized striatal samples (STR-array). We identified hundreds of differentially expressed genes (DEGs) and enhanced detection of MSN-specific DEGs by comparing the results of the FACS-array with those of the STR-array. The gene sets obtained included genes ubiquitously expressed in both MSNs and non-MSN cells of the brain and associated with transcriptional regulation and DNA damage responses. We proposed that the comparative gene expression approach using the FACS-array may be useful for uncovering the gene cascades affected in MSNs during HD pathogenesis.

    DOI: 10.1074/jbc.RA120.012983

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  • Proteomics-Based Approach Identifies Altered ER Domain Properties by ALS-Linked VAPB Mutation. 査読 国際誌

    Tomoyuki Yamanaka, Risa Nishiyama, Tomomi Shimogori, Nobuyuki Nukina

    Scientific reports   10 ( 1 )   7610 - 7610   2020年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    An ER transmembrane protein, vesicle-associated membrane protein-associated protein B (VAPB), binds to several organelle-resident membrane proteins to mediate ER-organelle tethering. Mutation in amyotrophic lateral sclerosis (ALS) induces protein misfolding and aggregation, leading to ER disorganization. Gain or loss of function is suggested for VAPB mutation, however comprehensive study focusing on VAPB-ER domain has yet been performed. We here conducted proteomic characterization of the ER containing VAPB and its ALS-linked P56S mutant. For this purpose, we first optimized the proteomics of different ER domains immuno-isolated from cultured cells, and identified ER sheet- and tubule-specific proteomes. By using these as references, we found that VAPB-ER proteome had intermediate ER domain properties but its tubular property was specifically decreased by its mutation. Biochemical, immunofluorescence and proximity ligation assays suggested this was mediated by delocalization of VAPB from ER tubules. The VAPB-ER proteomics further suggested reduced incorporation of multiple proteins located in different organelles, which was confirmed by proximity ligation assay. Taken together, our proteomics-based approach indicates altered ER domain properties and impaired ER-organelle tethering by VAPB mutation.

    DOI: 10.1038/s41598-020-64517-z

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  • Preserved proteinase K-resistant core after amplification of alpha-synuclein aggregates: Implication to disease-related structural study. 査読 国際誌

    Saki Yoshinaga, Tomoyuki Yamanaka, Haruko Miyazaki, Ayami Okuzumi, Akiko Hiyama, Shigeo Murayama, Nobuyuki Nukina

    Biochemical and biophysical research communications   522 ( 3 )   655 - 661   2020年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Many pathological proteins related to neurodegenerative diseases are misfolded, aggregating to form amyloid fibrils during pathogenesis. One of the pathological proteins, alpha-synuclein (α-syn), accumulates in the brains of Parkinson disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA), which are designated as synucleinopathies. Recently, structural properties of abnormal accumulated proteins are suggested to determine the disease phenotype. However, the biochemical and structural characteristics of those accumulated proteins are still poorly understood. We previously reported the sequence and seed-structure-dependent polymorphic fibrils of α-syn and the polymorphism was identified by proteinase K-resistant cores determined by mass spectrometry (MS) analysis. In this study, we applied this method to analyze α-syn aggregates of MSA and DLB. To perform MS analysis on proteinase K-resistant cores, we first performed amplification of α-syn aggregates by seeding reaction and protein misfolding cyclic amplification (PMCA) to obtain a sufficient amount of aggregates. Using SDS insoluble fraction of the disease brain, we successfully amplified enough α-syn aggregates for MS analysis. We differentiated between mouse and human α-syn aggregates by MS analysis on proteinase K-resistant cores of the aggregates before and after amplification. The results suggest that structural properties of amplified α-syn fibrils are preserved after PMCA and these methods can be applicable in the study of pathological proteins of the neurodegenerative disorders.

    DOI: 10.1016/j.bbrc.2019.11.142

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  • Non-coding RNA Neat1 and Abhd11os expressions are dysregulated in medium spiny neurons of Huntington disease model mice. 査読 国際誌

    Hongsun Park, Haruko Miyazaki, Tomoyuki Yamanaka, Nobuyuki Nukina

    Neuroscience research   147   58 - 63   2019年10月

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    記述言語:英語  

    Huntington Disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the exon1 of huntingtin gene (HTT). The mutant HTT affects the transcriptional profile of neurons by disrupting the activities of transcriptional machinery and alters expression of many genes. In this study, we identified dysregulated non-coding RNAs (ncRNAs) in medium spiny neurons of 4-week-old HD model mouse. Also, we observed the intracellular localizations of Abhd11os and Neat1 ncRNAs by ViewRNA in situ hybridization, which could provide more precise detection, suggesting that it is a useful method to investigate the expression changes of genes with low expression levels.

    DOI: 10.1016/j.neures.2018.10.013

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  • Sequence- and seed-structure-dependent polymorphic fibrils of alpha-synuclein. 査読 国際誌

    Goki Tanaka, Tomoyuki Yamanaka, Yoshiaki Furukawa, Naoko Kajimura, Kaoru Mitsuoka, Nobuyuki Nukina

    Biochimica et biophysica acta. Molecular basis of disease   1865 ( 6 )   1410 - 1420   2019年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Synucleinopathies comprise a diverse group of neurodegenerative diseases including Parkinson's disease (PD), dementia with Lewy bodies, and multiple system atrophy. These share a common pathological feature, the deposition of alpha-synuclein (a-syn) in neurons or oligodendroglia. A-syn is highly conserved in vertebrates, but the primary sequence of mouse a-syn differs from that of human at seven positions. However, structural differences of their aggregates remain to be fully characterized. In this study, we found that human and mouse a-syn aggregated in vitro formed morphologically distinct amyloid fibrils exhibiting twisted and straight structures, respectively. Furthermore, we identified different protease-resistant core regions, long and short, in human and mouse a-syn aggregates. Interestingly, among the seven unconserved amino acids, only A53T substitution, one of the familial PD mutations, was responsible for structural conversion to the straight-type. Finally, we checked whether the structural differences are transmissible by seeding and found that human a-syn seeded with A53T aggregates formed straight-type fibrils with short protease-resistant cores. These results suggest that a-syn aggregates form sequence-dependent polymorphic fibrils upon spontaneous aggregation but become seed structure-dependent upon seeding.

    DOI: 10.1016/j.bbadis.2019.02.013

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  • Biochemical and morphological classification of disease-associated alpha-synuclein mutants aggregates. 査読 国際誌

    Goki Tanaka, Tomoyuki Yamanaka, Yoshiaki Furukawa, Naoko Kajimura, Kaoru Mitsuoka, Nobuyuki Nukina

    Biochemical and biophysical research communications   508 ( 3 )   729 - 734   2019年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Alpha-synuclein (a-syn) aggregation in brain is implicated in several synucleinopathies, including Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). Until date, at least six disease-associated mutations in a-syn (namely A30P, E46K, H50Q, G51D, A53T, and A53E) are known to cause dominantly inherited familial forms of synucleinopathies. Previous studies using recombinant proteins have reported that a subset of disease-associated mutants show higher aggregation propensities and form spectroscopically distinguishable aggregates compared to wild-type (WT). However, morphological and biochemical comparison of the aggregates for all disease-associated a-syn mutants have not yet been performed. In this study, we performed electron microscopic examination, guanidinium hydrochloride (GdnHCl) denaturation, and protease digestion to classify the aggregates from their respective point mutations. Using electron microscopy we observed variations of amyloid fibrillar morphologies among the aggregates of a-syn mutants, mainly categorized into two groups: twisted fibrils observed for both WT and E46K while straight fibrils for the other mutants. GdnHCl denaturation experiments revealed the a-syn mutants except for E46K were more resistant than WT against the denaturation. Mass spectrometry analysis of protease-treated aggregates showed a variety of protease-resistant cores, which may correspond to their morphological properties. The difference of their properties could be implicated in the clinicopathological difference of synucleinopathies with those mutations.

    DOI: 10.1016/j.bbrc.2018.11.200

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  • Rapid dissemination of alpha-synuclein seeds through neural circuits in an in-vivo prion-like seeding experiment. 査読 国際誌

    Ayami Okuzumi, Masaru Kurosawa, Taku Hatano, Masashi Takanashi, Shuuko Nojiri, Takeshi Fukuhara, Tomoyuki Yamanaka, Haruko Miyazaki, Saki Yoshinaga, Yoshiaki Furukawa, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    Acta neuropathologica communications   6 ( 1 )   96 - 96   2018年9月

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    記述言語:英語  

    Accumulating evidence suggests that the lesions of Parkinson's disease (PD) expand due to transneuronal spreading of fibrils composed of misfolded alpha-synuclein (a-syn), over the course of 5-10 years. However, the precise mechanisms and the processes underlying the spread of these fibril seeds have not been clarified in vivo. Here, we investigated the speed of a-syn transmission, which has not been a focus of previous a-syn transmission experiments, and whether a-syn pathologies spread in a neural circuit-dependent manner in the mouse brain. We injected a-syn preformed fibrils (PFFs), which are seeds for the propagation of a-syn deposits, either before or after callosotomy, to disconnect bilateral hemispheric connections. In mice that underwent callosotomy before the injection, the propagation of a-syn pathology to the contralateral hemisphere was clearly reduced. In contrast, mice that underwent callosotomy 24 h after a-syn PFFs injection showed a-syn pathology similar to that seen in mice without callosotomy. These results suggest that a-syn seeds are rapidly disseminated through neuronal circuits immediately after seed injection, in a prion-like seeding experiment in vivo, although it is believed that clinical a-syn pathologies take years to spread throughout the brain. In addition, we found that botulinum toxin B blocked the transsynaptic transmission of a-syn seeds by specifically inactivating the synaptic vesicle fusion machinery. This study offers a novel concept regarding a-syn propagation, based on the Braak hypothesis, and also cautions that experimental transmission systems may be examining a unique type of transmission, which differs from the clinical disease state.

    DOI: 10.1186/s40478-018-0587-0

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  • ER dynamics and derangement in neurological diseases 査読

    Tomoyuki Yamanaka, Nobuyuki Nukina

    Frontiers in Neuroscience   12   91   2018年2月

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    記述言語:英語   出版者・発行元:Frontiers Media S.A.  

    The endoplasmic reticulum (ER) is a morphologically dynamic organelle containing different membrane subdomains with distinct cellular functions. Numerous observations have revealed that ER stress response induced by disturbed ER homeostasis is linked to various neurological/neurodegenerative disorders. In contrast, recent findings unveil that ER structural derangements are linked to the progression of several neurological diseases. The derangements involve two distinct, and likely opposing pathways. One is dysfunction of ER dynamics machinery, leading to disruption of ER network organization. Another one is facilitation of pre-existing machinery, leading to generation of markedly-ordered de novo membranous structure. Restoring the ER network can be the effective way toward the cure of ER-deranged neurological disorders.

    DOI: 10.3389/fnins.2018.00091

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  • Reappraisal of VAChT-Cre: Preference in slow motor neurons innervating type I or IIa muscle fibers 査読

    Hidemi Misawa, Daijiro Inomata, Miseri Kikuchi, Sae Maruyama, Yasuhiro Moriwaki, Takashi Okuda, Nobuyuki Nukina, Tomoyuki Yamanaka

    GENESIS   54 ( 11 )   568 - 572   2016年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    VAChT-Cre. Fast and VAChT-Cre. Slow mice selectively express Cre recombinase in approximately one half of postnatal somatic motor neurons. The mouse lines have been used in various studies with selective genetic modifications in adult motor neurons. In the present study, we crossed VAChT-Cre lines with a reporter line, CAG-Syp/tdTomato, in which synaptophysin-tdTomato fusion proteins are efficiently sorted to axon terminals, making it possible to label both cell bodies and axon terminals of motor neurons. In the mice, Syp/tdTomato fluorescence preferentially co-localized with osteopontin, a recently discovered motor neuron marker for slow-twitch fatigue-resistant (S) and fast-twitch fatigue-resistant (FR) types. The fluorescence did not preferentially co-localize with matrix metalloproteinase-9, a marker for fast-twitch fatigable (FF) motor neurons. In the neuromuscular junctions, Syp/tdTomato fluorescence was detected mainly in motor nerve terminals that innervate type I or IIa muscle fibers. These results suggest that the VAChT-Cre lines are Cre-drivers that have selectivity in S and FR motor neurons. In order to avoid confusion, we have changed the mouse line names from VAChT-Cre. Fast and VAChT-Cre. Slow to VAChT-Cre. Early and VAChT-Cre. Late, respectively. The mouse lines will be useful tools to study slow-type motor neurons, in relation to physiology and pathology.

    DOI: 10.1002/dvg.22979

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  • Differential roles of NF-Y transcription factor in ER chaperone expression and neuronal maintenance in the CNS 査読

    Tomoyuki Yamanaka, Asako Tosaki, Haruko Miyazaki, Masaru Kurosawa, Masato Koike, Yasuo Uchiyama, Sankar N. Maity, Hidemi Misawa, Ryosuke Takahashi, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    SCIENTIFIC REPORTS   6   34575   2016年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    The mammalian central nervous system (CNS) contains various types of neurons with different neuronal functions. In contrast to established roles of cell type-specific transcription factors on neuronal specification and maintenance, whether ubiquitous transcription factors have conserved or differential neuronal function remains uncertain. Here, we revealed that inactivation of a ubiquitous factor NF-Y in different sets of neurons resulted in cell type-specific neuropathologies and gene downregulation in mouse CNS. In striatal and cerebellar neurons, NF-Y inactivation led to ubiquitin/p62 pathologies with downregulation of an endoplasmic reticulum (ER) chaperone Grp94, as we previously observed by NF-Y deletion in cortical neurons. In contrast, NF-Y inactivation in motor neurons induced neuronal loss without obvious protein deposition. Detailed analysis clarified downregulation of another ER chaperone Grp78 in addition to Grp94 in motor neurons, and knockdown of both ER chaperones in motor neurons recapitulated the pathology observed after NF-Y inactivation. Finally, additional downregulation of Grp78 in striatal neurons suppressed ubiquitin accumulation induced by NF-Y inactivation, implying that selective ER chaperone downregulation mediates different neuropathologies. Our data suggest distinct roles of NF-Y in protein homeostasis and neuronal maintenance in the CNS by differential regulation of ER chaperone expression.

    DOI: 10.1038/srep34575

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  • Genome-wide analyses in neuronal cells reveal that upstream transcription factors regulate lysosomal gene expression 査読

    Tomoyuki Yamanaka, Asako Tosaki, Masaru Kurosawa, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    FEBS JOURNAL   283 ( 6 )   1077 - 1087   2016年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL  

    The upstream transcription factors (USFs) USF1 and USF2 are ubiquitously expressed transcription factors that are characterized by a conserved basic helix-loop-helix/leucine zipper DNA-binding domain. They form homo-or heterodimers, and recognize E-box motifs to modulate gene expression. They are known to regulate diverse cellular functions, including the cell cycle, immune responses and glucose/lipid metabolism, but their roles in neuronal cells remain to be clarified. Here, we performed chromatin immunoprecipitation of USF1 from mouse brain cortex. Subsequent promoter array analysis (ChIP-chip) indicated that USF1 exclusively bound to the CACGTG E-box motifs in the proximal promoter regions. Importantly, functional annotation of the USF1-binding targets revealed an enrichment of genes related to lysosomal functions. Gene expression array analysis using a neuronal cell line subsequently revealed that knockdown of USFs de-regulated lysosomal gene expression. Altered expression was validated by quantitative RT-PCR, supporting the conclusion that USFs regulate lysosomal gene expression. Furthermore, USF knockdown slightly increased LysoTracker Red staining, implying a role for USFs in modulating lysosomal homeostasis. Together, our comprehensive genome-scale analyses identified lysosomal genes as targets of USFs in neuronal cells, suggesting a potential additional pathway of lysosomal regulation.

    DOI: 10.1111/febs.13650

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  • Large-Scale RNA Interference Screening in Mammalian Cells Identifies Novel Regulators of Mutant Huntingtin Aggregation 査読

    Tomoyuki Yamanaka, Hon Kit Wong, Asako Tosaki, Peter O. Bauer, Koji Wada, Masaru Kurosawa, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    PLOS ONE   9 ( 4 )   e93891   2014年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PUBLIC LIBRARY SCIENCE  

    In polyglutamine (polyQ) diseases including Huntington's disease (HD), mutant proteins containing expanded polyQ stretch form aggregates in neurons. Genetic or RNAi screenings in yeast, C. elegans or Drosophila have identified multiple genes modifying polyQ aggregation, a few of which are confirmed effective in mammals. However, the overall molecular mechanism underlying polyQ protein aggregation in mammalian cells still remains obscure. We here perform RNAi screening in mouse neuro2a cells to identify mammalian modifiers for aggregation of mutant huntingtin, a causative protein of HD. By systematic cell transfection and automated cell image analysis, we screen similar to 12000 shRNA clones and identify 111 shRNAs that either suppress or enhance mutant huntingtin aggregation, without altering its gene expression. Classification of the shRNA-targets suggests that genes with various cellular functions such as gene transcription and protein phosphorylation are involved in modifying the aggregation. Subsequent analysis suggests that, in addition to the aggregation-modifiers sensitive to proteasome inhibition, some of them, such as a transcription factor Tcf20, and kinases Csnk1d and Pik3c2a, are insensitive to it. As for Tcf20, which contains polyQ stretches at N-terminus, its binding to mutant huntingtin aggregates is observed in neuro2a cells and in HD model mouse neurons. Notably, except Pik3c2a, the rest of the modifiers identified here are novel. Thus, our first large-scale RNAi screening in mammalian system identifies previously undescribed genetic players that regulate mutant huntingtin aggregation by several, possibly mammalian-specific mechanisms.

    DOI: 10.1371/journal.pone.0093891

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  • NF-Y inactivation causes atypical neurodegeneration characterized by ubiquitin and p62 accumulation and endoplasmic reticulum disorganization 査読

    Tomoyuki Yamanaka, Asako Tosaki, Masaru Kurosawa, Gen Matsumoto, Masato Koike, Yasuo Uchiyama, Sankar N. Maity, Tomomi Shimogori, Nobutaka Hattori, Nobuyuki Nukina

    NATURE COMMUNICATIONS   5   3354   2014年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    Nuclear transcription factor-Y (NF-Y), a key regulator of cell-cycle progression, often loses its activity during differentiation into nonproliferative cells. In contrast, NF-Y is still active in mature, differentiated neurons, although its neuronal significance remains obscure. Here we show that conditional deletion of the subunit NF-YA in postmitotic mouse neurons induces progressive neurodegeneration with distinctive ubiquitin/p62 pathology; these proteins are not incorporated into filamentous inclusion but co-accumulated with insoluble membrane proteins broadly on endoplasmic reticulum (ER). The degeneration also accompanies drastic ER disorganization, that is, an aberrant increase in ribosome-free ER in the perinuclear region, without inducing ER stress response. We further perform chromatin immunoprecipitation and identify several NF-Y physiological targets including Grp94 potentially involved in ER disorganization. We propose that NF-Y is involved in a unique regulation mechanism of ER organization in mature neurons and its disruption causes previously undescribed novel neuropathology accompanying abnormal ubiquitin/p62 accumulation.

    DOI: 10.1038/ncomms4354

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  • Loss of aPKCλ in differentiated neurons disrupts the polarity complex but does not induce obvious neuronal loss or disorientation in mouse brains. 査読

    Yamanaka T, Tosaki A, Kurosawa M, Akimoto K, Hirose T, Ohno S, Hattori N, Nukina N

    PloS one   8 ( 12 )   e84036   2013年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:12  

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  • Mutant huntingtin fragment selectively suppresses Brn-2 POU domain transcription factor to mediate hypothalamic cell dysfunction 査読

    Tomoyuki Yamanaka, Asako Tosaki, Haruko Miyazaki, Masaru Kurosawa, Yoshiaki Furukawa, Mizuki Yamada, Nobuyuki Nukina

    HUMAN MOLECULAR GENETICS   19 ( 11 )   2099 - 2112   2010年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:OXFORD UNIV PRESS  

    In polyglutamine diseases including Huntington's disease (HD), mutant proteins containing expanded polyglutamine stretches form nuclear aggregates in neurons. Although analysis of their disease models suggested a significance of transcriptional dysregulation in these diseases, how it mediates the specific neuronal cell dysfunction remains obscure. Here we performed a comprehensive analysis of altered DNA binding of multiple transcription factors using R6/2 HD model mice brains that express an N-terminal fragment of mutant huntingtin (mutant Nhtt). We found a reduction of DNA binding of Brn-2, a POU domain transcription factor involved in differentiation and function of hypothalamic neurosecretory neurons. We provide evidence supporting that Brn-2 loses its function through two pathways, its sequestration by mutant Nhtt and its reduced transcription, leading to reduced expression of hypothalamic neuropeptides. In contrast to Brn-2, its functionally related protein, Brn-1, was not sequestered by mutant Nhtt but was upregulated in R6/2 brain, except in hypothalamus. Our data indicate that functional suppression of Brn-2 together with a region-specific lack of compensation by Brn-1 mediates hypothalamic cell dysfunction by mutant Nhtt.

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  • Transcription factor sequestration by polyglutamine proteins.

    Yamanaka T, Nukina N

    Methods in molecular biology (Clifton, N.J.)   648   215 - 229   2010年

  • Intracellular polarity protein PAR-1 regulates extracellular laminin assembly by regulating the dystroglycan complex 査読

    Maki Masuda-Hirata, Atsushi Suzuki, Yoshiko Amano, Kazunari Yamashita, Mariko Ide, Tomoyuki Yamanaka, Michihiro Sakai, Michihiro Imamura, Shigeo Ohno

    GENES TO CELLS   14 ( 7 )   835 - 850   2009年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:WILEY-BLACKWELL PUBLISHING, INC  

    Cell polarity depends on extrinsic spatial cues and intrinsic polarity proteins including PAR-aPKC proteins. In mammalian epithelial cells, cell-cell contacts provide spatial cues that activate the aPKC-PAR-3-PAR-6 complex to establish the landmark of the initial cellular asymmetry. PAR-1, a downstream target of the aPKC-PAR-3-PAR-6 complex, mediates further development of the apical and basolateral membrane domains. However, the relationships between the PAR-aPKC proteins and other extrinsic spatial cues provided by the extracellular matrix (ECM) remain unclear. Here, we show that PAR-1 colocalizes with laminin receptors and is required for the assembly of extracellular laminin on the basal surface of epithelial cells. Furthermore, PAR-1 regulates the basolateral localization of the dystroglycan (DG) complex, one of the laminin receptors essential for basement membrane formation. We also show that PAR-1 interacts with the DG complex and is required for the formation of a functional DG complex. These results reveal the presence of a novel inside-out pathway in which an intracellular polarity protein regulates the ECM organization required for epithelial cell polarity and tissue morphogenesis.

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  • Interaction between PAR-3 and the aPKC-PAR-6 complex is indispensable for apical domain development of epithelial cells 査読

    Yosuke Horikoshi, Atsushi Suzuki, Tomoyuki Yamanaka, Kazunori Sasaki, Keiko Mizuno, Hajime Sawada, Shigenobu Yonemura, Shigeo Ohno

    JOURNAL OF CELL SCIENCE   122 ( 10 )   1595 - 1606   2009年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:COMPANY OF BIOLOGISTS LTD  

    The evolutionarily conserved polarity proteins PAR-3, atypical protein kinase C (aPKC) and PAR-6 critically regulate the apical membrane development required for epithelial organ development. However, the molecular mechanisms underlying their roles remain to be clarified. We demonstrate that PAR-3 knockdown in MDCK cells retards apical protein delivery to the plasma membrane, and eventually leads to mislocalized apical domain formation at intercellular regions in both two-dimensional and three-dimensional culture systems. The defects in PAR-3 knockdown cells are efficiently rescued by wild-type PAR-3, but not by a point mutant (S827/829A) that lacks the ability to interact with aPKC, indicating that formation of the PAR-3-aPKC-PAR-6 complex is essential for apical membrane development. This is in sharp contrast with tight junction maturation, which does not necessarily depend on the aPKC-PAR-3 interaction, and indicates that the two fundamental processes essential for epithelial polarity are differentially regulated by these polarity proteins. Importantly, highly depolarized cells accumulate aPKC and PAR-6, but not PAR-3, on apical protein-containing vacuoles, which become targeted to PAR-3-positive primordial cell-cell contact sites during the initial stage of the repolarization process. Therefore, formation of the PAR-3-aPKC-PAR-6 complex might be required for targeting of not only the aPKC-PAR-6 complex but also of apical protein carrier vesicles to primordial junction structures.

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  • Intranuclear Degradation of Polyglutamine Aggregates by the Ubiquitin-Proteasome System 査読

    Atsushi Iwata, Yu Nagashima, Lumine Matsumoto, Takahiro Suzuki, Tomoyuki Yamanaka, Hidetoshi Date, Ken Deoka, Nobuyuki Nukina, Shoji Tsuji

    JOURNAL OF BIOLOGICAL CHEMISTRY   284 ( 15 )   9796 - 9803   2009年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    Huntington disease and its related autosomal-dominant polyglutamine (pQ) neurodegenerative diseases are characterized by intraneuronal accumulation of protein aggregates. Studies on protein aggregates have revealed the importance of the ubiquitin-proteasome system as the front line of protein quality control (PQC) machinery against aberrant proteins. Recently, we have shown that the autophagy-lysosomal system is also involved in cytoplasmic aggregate degradation, but the nucleus lacked this activity. Consequently, the nucleus relies entirely on the ubiquitin-proteasome system for PQC. According to previous studies, nuclear aggregates possess a higher cellular toxicity than do their cytoplasmic counterparts, however degradation kinetics of nuclear aggregates have been poorly understood. Here we show that nuclear ubiquitin ligases San1p and UHRF-2 each enhance nuclear pQ aggregate degradation and rescued pQ-induced cytotoxicity in cultured cells and primary neurons. Moreover, UHRF-2 is associated with nuclear inclusion bodies in vitro and in vivo. Our data suggest that UHRF-2 is an essential molecule for nuclear pQ degradation as a component of nuclear PQC machinery in mammalian cells.

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  • Role of Lgl/Dlg/Scribble in the regulation of epithelial junction, polarity and growth. 査読 国際誌

    Tomoyuki Yamanaka, Shigeo Ohno

    Frontiers in bioscience : a journal and virtual library   13   6693 - 707   2008年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:FRONTIERS IN BIOSCIENCE INC  

    Genetic studies in Drosophila have revealed that three tumor suppressors, Discs large (Dlg), Scribble (Scrib) and Lethal giant larvae (Lgl), which localize to the basolateral region of epithelial cells, cooperatively regulate cell polarity, junction formation and cell growth in epithelial cells. Subsequent studies in Drosophila, vertebrates and C. elegans have shown the evolutionary conservation of some of their functions in epithelial cells. Also, these studies revealed the importance of antagonistic interactions between these tumor suppressors and apical polarity regulators such as Crumbs and aPKC for the establishment of apical-basal polarity with organized cell-cell junctions and regulation of cell growth in epithelial cells.

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  • Genetic impairment of autophagy intensifies expanded polyglutamine toxicity in Caenorhabditis elegans 査読

    Liakot A. Khan, Tomoyuki Yamanaka, Nobuyuki Nukina

    BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS   368 ( 3 )   729 - 735   2008年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACADEMIC PRESS INC ELSEVIER SCIENCE  

    Neuronal homeostasis requires a balance between anabolic and catabolic processes. Eukaryotic cells use two distinct systems for the degradation of unused proteins: the ubiquitin-proteasome system and the autophagic system. The autophagic system is also necessary for the degradation of bulk amounts of proteins and organelles. We have searched for new autophagy-related genes in the Caenorhabditis elegans genome and investigated their role in a polyglutamine (polyQ) disease model. Here, we have shown that inactivation of these genes intensified the toxicity of expanded polyQ in C elegans neurons and muscles, and at the same time inactivation of CeTor reduced the polyQ toxicity. (c) 2008 Elsevier Inc. All rights reserved.

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  • Mutant Huntingtin reduces HSP70 expression through the sequestration of NF-Y transcription factor 査読

    Tomoyuki Yamanaka, Haruko Miyazaki, Fumitaka Oyama, Masaru Kurosawa, Chika Washizu, Hiroshi Doi, Nobuyuki Nukina

    EMBO JOURNAL   27 ( 6 )   827 - 839   2008年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:NATURE PUBLISHING GROUP  

    In Huntington's disease (HD), mutant Huntingtin, which contains expanded polyglutamine stretches, forms nuclear aggregates in neurons. The interactions of several transcriptional factors with mutant Huntingtin, as well as altered expression of many genes in HD models, imply the involvement of transcriptional dysregulation in the HD pathological process. The precise mechanism remains obscure, however. Here, we show that mutant Huntingtin aggregates interact with the components of the NF-Y transcriptional factor in vitro and in HD model mouse brain. An electrophoretic mobility shift assay using HD model mouse brain lysates showed reduction in NF-Y binding to the promoter region of HSP70, one of the NF-Y targets. RT-PCR analysis revealed reduced HSP70 expression in these brains. We further clarified the importance of NF-Y for HSP70 transcription in cultured neurons. These data indicate that mutant Huntingtin sequesters NF-Y, leading to the reduction of HSP70 gene expression in HD model mice brain. Because suppressive roles of HSP70 on the HD pathological process have been shown in several HD models, NF-Y could be an important target of mutant Huntingtin.

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  • Lgl mediates apical domain disassembly by suppressing the PAR-3-aPKC-PAR-6 complex to orient apical membrane polarity 査読

    T Yamanaka, Y Horikoshi, N Izumi, A Suzuki, K Mizuno, S Ohno

    JOURNAL OF CELL SCIENCE   119 ( 10 )   2107 - 2118   2006年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:COMPANY OF BIOLOGISTS LTD  

    The basolateral tumor suppressor protein Lgl is important for the regulation of epithelial cell polarity and tissue morphology. Recent studies have shown a physical and functional interaction of Lgl with another polarity-regulating protein machinery, the apical PAR3-aPKC-PAR-6 complex, in epithelial cells. However, the mechanism of Lgl-mediated regulation of epithelial cell polarity remains obscure. By an siRNA method, we here show that endogenous Lgl is required for the disassembly of apical membrane domains in depolarizing MDCK cells induced by Ca2+ depletion. Importantly, this Lgl function is mediated by the suppression of the apical PAR3-aPKC-PAR-6 complex activity. Analysis using 2D- or 3D-cultured cells in collagen gel suggests the importance of this suppressive regulation of Lgl on the collagen-mediated re-establishment of apical membrane domains and lumen formation. These results indicate that basolateral Lgl plays a crucial role in the disassembly of apical membrane domains to induce the orientation of apical membrane polarity, which is mediated by the suppression of apical PAR-3-aPKC-PAR-6 complex activity.

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  • Direct binding of lg12 to LGN during mitosis and its requirement for normal cell division 査読

    M Yasumi, T Sakisaka, T Hoshino, T Kimura, Y Sakamoto, T Yamanaka, S Ohno, Y Takai

    JOURNAL OF BIOLOGICAL CHEMISTRY   280 ( 8 )   6761 - 6765   2005年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

    The Drosophila tumor suppressor protein lethal (2) giant larvae (1(2)gl) is involved in asymmetric cell division during development and epithelial cell polarity through interaction with the aPKC-Par-6 complex. We showed here that Lgl2, a mammalian homolog of 1(2)gl, directly bound to LGN, a mammalian homolog of Partner of inscuteable in HEK293 cells. The C-terminal tail of Lgl2 bound to LGN with a K-d value of about 56 nm. Endogenous Lgl2 formed a complex with aPKC, Par-6, and LGN. This complex formation was enhanced in metaphase of the synchronized cells by treatment with thymidine and nocodazole. Immunofluoreseence staining of the complex was the strongest at the cell periphery of the metaphase cells. Overexpression of the C-terminal tail of Lgl2 induced mis-localization of the nuclear mitotic apparatus protein NuMA and disorganization of the mitotic spindle during mitosis, eventually causing formation of multiple micronuclei. Knockdown of endogenous Lgl (Lgl1 and Lgl2) also induced disorganization of the mitotic spindle, thereby causing formation of multiple micronuclei. The binding between Lg12 and LGN played a role in the mitotic spindle organization through regulating formation of the LGN-NuMA complex. These results indicate that Lg12 forms a Lgl2(.)Par-6(.)aPKC(.)LGN complex, which responds to mitotic signaling to establish normal cell division.

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  • aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity 査読

    A Suzuki, M Hirata, K Kamimura, R Maniwa, T Yamanaka, K Mizuno, M Kishikawa, H Hirose, Y Amano, N Izumi, Y Miwa, S Ohno

    CURRENT BIOLOGY   14 ( 16 )   1425 - 1435   2004年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CELL PRESS  

    Background: aPKC and PARA are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions(TJs) and playsan indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. On the other hand, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/ MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC.
    Results: We demonstrate that PAR-1b is essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. On the other hand, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development.
    Conclusions: These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity.

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  • Mammalian LgI forms a protein complex with PAR-6 and aPKC independently of PAR-3 to regulate epithelial cell polarity 査読

    T Yamanaka, Y Horikoshi, Y Sugiyama, C Ishiyama, A Suzuki, T Hirose, A Iwamatsu, A Shinohara, S Ohno

    CURRENT BIOLOGY   13 ( 9 )   734 - 743   2003年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:CELL PRESS  

    Background: Epithelial cells have apicobasal polarity and an asymmetric junctional complex that provides the bases for development and tissue maintenance. In both vertebrates and invertebrates, the evolutionarily conserved protein complex, PAR-6/aPKC/PAR-3, localizes to the subapical region and plays critical roles in the establishment of a junctional complex and cell polarity. In Drosophila, another set of proteins called tumor suppressors, such as LgI, which localize separately to the basolateral membrane domain but genetically interact with the subapical proteins, also contribute to the establishment of cell polarity. However, how physically separated proteins interact remains to be clarified.
    Results: We show that mammalian LgI competes for PAR-3 in forming an independent complex with PAR-6/ aPKC. During cell polarization, mLgI initially colocalizes with PAR-6/aPKC at the cell-cell contact region and is phosphorylated by aPKC, followed by segregation from apical PAR-6/aPKC to the basolateral membrane after cells are polarized. Overexpression studies establish that increased amounts of the mLgI/PAR-6/aPKC complex suppress the formation of epithelial junctions; this contrasts with the previous observation that the complex containing PAR-3 promotes it.
    Conclusions: These results indicate that PAR-6/aPKC selectively interacts with either mLgI or PAR-3 under the control of aPKC activity to regulate epithelial cell polarity.

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  • Involvement of ASIP/PAR-3 in the promotion of epithelial tight junction formation 査読

    T Hirose, Y Izumi, Y Nagashima, Y Tamai-Nagai, H Kurihara, T Sakai, Y Suzuki, T Yamanaka, A Suzuki, K Mizuno, S Ohno

    JOURNAL OF CELL SCIENCE   115 ( 12 )   2485 - 2495   2002年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:COMPANY OF BIOLOGISTS LTD  

    The mammalian protein ASIP/PAR-3 interacts with atypical protein kinase C isotypes (aPKC) and shows overall sequence similarity to the invertebrate proteins C. elegans PAR-3 and Drosophila Bazooka, which are crucial for the establishment of polarity in various cells. The physical interaction between ASIP/PAR-3 and aPKC is also conserved in C. elegans PAR-3 and PKC-3 and in Drosophila Bazooka and DaPKC. In mammals, ASIP/PAR-3 colocalizes with aPKC and concentrates at the tight junctions of epithelial cells, but the biological meaning of ASIP/PAR-3 in tight junctions remains to be clarified. In the present study, we show that ASIP/PAR-3 staining distributes to the subapical domain of epithelial cell-cell junctions, including epithelial cells with less-developed tight junctions, in clear contrast with ZO-1, another tight-junction-associated protein, the staining of which is stronger in cells with well-developed tight junctions. Consistently, immunogold electron microscopy revealed that ASIP/PAR-3 concentrates at the apical edge of tight junctions, whereas ZO-1 distributes alongside tight junctions. To clarify the meaning of this characteristic localization of ASIP, we analyzed the effects of overexpressed ASIP/PAR-3 on tight junction formation in cultured epithelial MDCK cells. The induced overexpression of ASIP/PAR-3, but not its deletion mutant lacking the aPKC-binding sequence, promotes cell-cell contact-induced tight junction formation in MDCK cells when evaluated on the basis of transepithelial electrical resistance and occludin insolubilization. The significance of the aPKC-binding sequence in tight junction formation is also supported by the finding that the conserved PKC-phosphorylation site within this sequence, ASIP-Ser827, is phosphorylated at the most apical tip of cell-cell contacts during the initial phase of tight junction formation in MDCK cells. Together, our present data suggest that ASIP/PAR-3 regulates epithelial tight junction formation positively through interaction with aPKC.

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  • PAR-6 regulates aPKC activity in a novel way and mediates cell-cell contact-induced formation of the epithelial junctional complex. 査読

    Yamanaka T, Horikoshi Y, Suzuki A, Sugiyama Y, Kitamura K, Maniwa R, Nagai Y, Yamashita A, Hirose T, Ishikawa H, Ohno S

    Genes to cells : devoted to molecular & cellular mechanisms   6 ( 8 )   721 - 731   2001年8月

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  • Atypical protein kinase C is involved in the evolutionarily conserved PAR protein complex and plays a critical role in establishing epithelia-specific junctional structures 査読

    A Suzuki, T Yamanaka, T Hirose, N Manabe, K Mizuno, M Shimizu, K Akimoto, Y Izumi, T Ohnishi, S Ohno

    JOURNAL OF CELL BIOLOGY   152 ( 6 )   1183 - 1196   2001年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ROCKEFELLER UNIV PRESS  

    We have previously shown that during early Caenorhabditis elegans embryogenesis PKC-3, a C. elegans atypical PKC (aPKC), plays critical roles in the establishment of cell polarity required for subsequent asymmetric cleavage by interacting with PAR-3 [Tabuse, Y., Y. Izumi, F. Piano, K.J. Kemphues, J. Miwa, and S. Ohno. 1998. Development (Camb.). 125:3607-3614]. Together with the fact that aPKC and a mammalian PAR-3 homologue, aPKC-specific interacting protein (ASIP), colocalize at the tight junctions of polarized epithelial cells (Izumi, Y., H. Hirose, Y. Tamai, S.-I. Hirai, Y. Nagashima, T. Fujimoto, Y. Tabuse, K.J. Kemphues, and S, Ohno. 1998. J. Cell Biol. 143:95-106), this suggests a ubiquitous role for aPKC in establishing cell polarity in multicellular organisms. Here. we show that the overexpression of a dominant-negative mutant of aPKC (aPKCkn) in MDCK II cells causes mislocalization of ASIP/PAR-3. Immunocytochemical analyses, as well as measurements of paracellular diffusion of ions or non-ionic solutes, demonstrate that the biogenesis of the tight junction structure itself is severely affected in aPKCkn-expressing cells. Furthermore, these cells show increased interdomain diffusion of fluorescent lipid and disruption of the polarized distribution of Na+, K+-ATPase, suggesting that epithelial cell surface polarity is severely impaired in these cells. On the other hand, we also found that aPKC associates not only with ASIP/PAR-3, but also with a mammalian homologue of C. elegans PAR-6 (mPAR-6). and thereby mediates the formation of an aPKC-ASIP/PAR-3-PAR-6 ternary complex that localizes to the apical junctional region of MDCK cells. These results indicate that aPKC is involved in the evolutionarily conserved PAR protein complex, and plays critical roles in the development of the junctional structures and apico-basal polarization of mammalian epithelial cells.

    DOI: 10.1083/jcb.152.6.1183

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  • Atypical protein kinase C lambda binds and regulates p70 S6 kinase 査読

    K Akimoto, M Nakaya, T Yamanaka, J Tanaka, S Matsuda, QP Weng, J Avruch, S Ohno

    BIOCHEMICAL JOURNAL   335   417 - 424   1998年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PORTLAND PRESS  

    p70 S6 kinase (p70 S6K) has been implicated in the regulation of cell cycle progression. However, the mechanism of its activation is not fully understood. In the present work, evidence is provided that an atypical protein kinase C (PKC) isotype, PKC lambda, is indispensable, but not sufficient, for the activation of p70 S6K. Both the regulatory and kinase domains of PKC lambda associate directly with p70 S6K. Overexpression of the kinase domain without kinase activity or the regulatory domain of PKC lambda results in the suppression of the serum-induced activation of p70 S6K. In addition, two types of dominant-negative mutants of PKC lambda, as well as a kinase-deficient mutant of p70 S6K, suppress serum-induced DNA synthesis and E2F activation. The overexpression of the active form of PKC lambda, however, fails to activate p70 S6K. These results suggest that PKC lambda is a mediator in the regulation of p70 S6K activity and plays an important role in cell cycle progression.

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MISC

  • マウス脳における脳梁離断を用いた線維状αsynucleinの伝播経路の検討

    奥住 文美, 波田野 琢, 黒澤 大, 山中 智行, 宮崎 晴子, 古川 良明, 服部 信孝, 貫名 信行

    パーキンソン病・運動障害疾患コングレスプログラム・抄録集   11回   68 - 68   2017年10月

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    記述言語:日本語   出版者・発行元:Movement Disorder Society of Japan (MDSJ)  

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  • NF-Y

    山中 智行

    脳内環境辞典(メディカルドゥ)   2017年3月

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  • 脳神経細胞の生存に関わる新規遺伝子の発見

    山中 智行

    理大科学フォーラム   2015年8月

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  • 転写因子 NF-Y の機能欠損マウスは新規のタンパク質蓄積病態を示す

    山中 智行

    包括脳ニュースレター   2015年3月

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  • 転写因子NF-Y欠損による新たな神経変性病態

    山中智行, 貫名信行

    医学のあゆみ   2014年10月

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  • 転写因子NF-Yの機能破壊はユビキチン・p62の蓄積、小胞体異常を伴う神経変性を誘導する

    山中 智行, 戸崎 麻子, 黒澤 大, 松本 弦, 小池 正人, 内山 安男, Maity Sankar N, 下郡 智美, 服部 信孝, 貫名 信行

    日本細胞生物学会大会講演要旨集   66回   135 - 135   2014年5月

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    記述言語:日本語   出版者・発行元:(一社)日本細胞生物学会  

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  • 転写因子NF-Yの機能破壊はユビキチン・p62の蓄積、小胞体異常を伴う神経変性を誘導する

    山中 智行, 戸崎 麻子, 黒澤 大, 松本 弦, 小池 正人, 内山 安男, Maity Sankar N, 下郡 智美, 服部 信孝, 貫名 信行

    日本薬学会年会要旨集   134年会 ( 3 )   87 - 87   2014年3月

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    記述言語:日本語   出版者・発行元:(公社)日本薬学会  

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  • Establishment of method for counting of polyglutamine aggregates by Array Scan reader and its application for shRNA high-throughput screening

    Tomoyuki Yamanaka, Asako Tosaki, Hon Kit Wong, Peter O. Bauer, Koji Wada, Nobuyuki Nukina

    NEUROSCIENCE RESEARCH   71   E192 - E192   2011年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:ELSEVIER IRELAND LTD  

    DOI: 10.1016/j.neures.2011.07.830

    Web of Science

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  • PAR‐3とaPKC‐PAR‐6との相互作用は,上皮細胞のアピカルドメインの形成に必要である。

    堀越洋輔, 鈴木厚, 山中智行, 佐々木和教, 水野恵子, 米村重信, 大野茂男

    レーザ顕微鏡研究会講演会抄録集   35th   47 - 50   2009年

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    記述言語:日本語  

    J-GLOBAL

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  • Mutant Huntingtin reduces HSP70 expression through the sequestration of NF-Y transcription factor

    Tomoyuki Yamanaka, Haruko Miyazaki, Fumitaka Oyama, Masaru Kurosawa, Chika Washizu, Hiroshi Doi, Nobuyuki Nukina

    NEUROSCIENCE RESEARCH   61   S45 - S45   2008年

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:ELSEVIER IRELAND LTD  

    Web of Science

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  • PAR‐3は,上皮細胞のアピカルドメインの形成に必要である。

    堀越洋輔, 山中智行, 泉奈津子, 水野恵子, 鈴木厚, 大野茂男

    日本分子生物学会年会講演要旨集   28th   632   2005年11月

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    記述言語:日本語  

    J-GLOBAL

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  • ほ乳類上皮細胞の極性制御におけるPAR‐3複合体,Lethal giant larvae複合体の機能解析

    山中智行, 堀越洋輔, 泉奈津子, 鈴木厚, 村松玲子, 三輪佳宏, 大野茂男

    日本分子生物学会年会プログラム・講演要旨集   27th   607   2004年11月

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    記述言語:日本語  

    J-GLOBAL

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  • PAR‐6のほ乳類上皮細胞極性形成における機能解析

    堀越洋輔, 山中智行, 鈴木厚, 大野茂男

    日本分子生物学会年会プログラム・講演要旨集   23rd   681   2000年11月

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    記述言語:日本語  

    J-GLOBAL

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  • aPKC群結合タンパク質の単離およびその生化学的解析

    田中純平, 秋本和憲, 山中智行, 中谷雅明, 吉田道彦, 広瀬智威, 鈴木厚, 田沼靖一, 大野茂男

    日本分子生物学会年会プログラム・講演要旨集   21st   550   1998年11月

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    記述言語:日本語  

    J-GLOBAL

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  • aPKCλはp70S6Kinaseに直接結合し,その活性制御に関与する

    秋本和憲, 中谷雅明, 山中智行, 田中純平, 上野芳夫, 田沼靖一, 武田健, AVRUCH J, 大野茂男

    日本分子生物学会年会プログラム・講演要旨集   20th   464   1997年12月

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    記述言語:日本語  

    J-GLOBAL

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  • p70S6キナーゼはaPKC/TRE経路に関与する

    中谷雅明, 秋本和憲, 山中智行, 田中純平, 松田伸一, 一原直昭, 大野茂男, KAZLAUSKAS A, AVRUCH J

    日本分子生物学会年会プログラム・講演要旨集   20th   465   1997年12月

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    記述言語:日本語  

    J-GLOBAL

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  • p70S6kinaseをリン酸化し,活性化するキナーゼ(S6KK)の解析

    山中智行, 秋本和憲, 田中純平, 宮本尚幸, 中谷雅明, 松田伸一, AVRUCH J, 上野芳夫, 大野茂男

    日本分子生物学会年会プログラム・講演要旨集   20th   462   1997年12月

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    記述言語:日本語  

    J-GLOBAL

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▶ 全件表示

共同研究・競争的資金等の研究

  • 変性神経細胞におけるダイナミックなクロマチン構造変化の同定

    研究課題/領域番号:18H02723  2018年4月 - 2022年3月

    日本学術振興会  科学研究費助成事業 基盤研究(B)  基盤研究(B)

    山中 智行, 貫名 信行, 岩田 淳

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    配分額:17420000円 ( 直接経費:13400000円 、 間接経費:4020000円 )

    ハンチントン病等のポリグルタミン病は、唯一核内に病因タンパク質の重合体(核内封入体)が形成される神経変性疾患である。神経細胞核内の機能異常がその疾患進行に関わっていると考えられ、実際、数千もの遺伝子が発現変化することが見出されている。これまでに、我々を含めた研究グループから、NF-Yなどいくつかの転写調節因子がハンチンチン封入体に取り込まれ、活性阻害されることが報告されている(Yamanaka T et al. EMBO J 2008, Hum Mol Genet 2010、PLoS ONE 2014 etc)。これらの結果は遺伝子発現異常による神経変性機構を一部説明するものであるが、これまで同定された約20個の転写調節因子では多大な遺伝子発現変化は説明できていない。すなわち、よりダイナミックな核内変化が存在すると予想される。本研究では、ハンチントン病の核内封入体を指標に変性神経細胞の細胞核を単離する方法を確立し、世界で初めて最新ゲノム技術を用いた変性神経細胞での包括的クロマチン構造解析を行うことにより、神経変性に関わる新規病態メカニズムを明らかにする。本年度は、ハンチントン病モデルマウス脳から神経細胞核を分離するため、まず、脳を懸濁し、NeuN抗体にて神経細胞核を標識し、セルソーターにて単離する方法を確立した。この細胞核よりゲノムDNAを抽出し、クロマチン免疫沈降やクロマチン構造解析に向け準備を進めている。一方、ハンチンチン凝集体に結合する転写因子NF-Yについて、その中枢神経系における機能解析をマウスを用いてさらに進め、Burke Neurological Institute Special Seminarにて口頭発表を行った(招待講演)。

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  • インタラクトームを基盤とした神経変性疾患の多様性を生み出す要因の解明

    研究課題/領域番号:17KT0131  2017年7月 - 2020年3月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    山中 智行

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    配分額:4550000円 ( 直接経費:3500000円 、 間接経費:1050000円 )

    神経変性疾患では、例え同じ病因タンパク質が凝集・蓄積しても異なる病態、症状を示すことが知られている。病態の複雑性を生み出す発症機構の解明は、疾患多様性に対応した予防・治療への喫緊の課題である。病因タンパク質の1つ、αシヌクレインは、パーキンソン病、多系統萎縮症やレビー小体型認知症で凝集・蓄積する(シヌクレイノパチー)。共通の凝集・蓄積タンパク質がどのようにして多様な病態が生じるかは未だ不明であるが、最近、αシヌクレインが異なるアミロイド線維を形成しうることが報告されており、これとシヌクレイノパチー多様性との関連が指摘されている。昨年度までに、ヒトαシヌクレインと共に、7つのアミノ酸残基が異なるマウスαシヌクレインに着目し解析を進め、電子顕微鏡解析により、これら凝集体は異なったアミロイド線維構造を有することを見出し、国内学会のシンポジウムで発表した。今年度は、さらに、質量分析解析により、これら凝集体が異なるプロテアーゼコア領域を持つことを見出すとともに、ヒトαシヌクレインの53位のアラニンをスレオニンに置換するだけで、アミロイド線維の構造変換には十分であることを明らかとした。さらに、家族性のシヌクレイノパチーに関連した変異体6つについても、電子顕微鏡解析、質量分析解析を行い、これらが異なる構造をもつ凝集体を形成することを明らかとした。以上をまとめ、Biochim Biophys Acta、Biochem Biophys Res Communに誌上発表した(共責任著者)。

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  • 神経細胞における新たな小胞体ストレス応答機構の解明

    研究課題/領域番号:15K06762  2015年4月 - 2019年3月

    日本学術振興会  科学研究費助成事業 基盤研究(C)  基盤研究(C)

    山中 智行, 下郡 智美, 貫名 信行, 小池 正人, 三澤 日出巳

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    配分額:4940000円 ( 直接経費:3800000円 、 間接経費:1140000円 )

    最近の解析から、UPR以外に、「小胞体凝集」という別の小胞体ストレス応答機構の存在が示唆されつつある。これは、小胞体膜タンパク質の変異や異常蓄積などによって誘導される。これまでに、我々は、転写因子NF-Yの脳錐体神経細胞での機能破壊が、小胞体凝集を伴うタンパク質蓄積病態を示すことを見出した(Nat Commun 2014)。本研究では、異なる神経細胞種を用いた比較解析から、小胞体凝集に関わるキーファクターを見出すと共に、RNA-seqやプロテオミクス解析をさらに行うことにより、小胞体凝集の病態メカニズムの全体像を見いだし、学会・論文等に発表した(Sci Rep 2016等)。

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  • 転写因子NF-Yを介した新たな神経維持・変性機構の解明

    研究課題/領域番号:24111553  2012年4月 - 2014年3月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    山中 智行

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    担当区分:研究代表者  資金種別:競争的資金

    配分額:11700000円 ( 直接経費:9000000円 、 間接経費:2700000円 )

    NF-Yは、NF-YA, -YB, -YCの3者複合体からなる転写因子であり、プロモーター領域のCCAATモチーフに結合し、遺伝子発現を制御している。そのノックアウトマウス・繊維芽細胞を用いた解析から、NF-Yが細胞周期制御を介した細胞増殖に必須であること、その活性は非分裂細胞へと分化する際に消失することが知られている。一方で、最近我々は、NF-Yが分化した非分裂の神経細胞でも活性があること、また、神経変性疾患であるハンチントン病で、その活性が低下することを明らかとした。これらのことから、NF-Yは分化した神経細胞でも、その維持・機能になんらかの役割を担っていると期待される。
    本研究ではNF-YAの脳神経細胞特異的ノックアウトが、グリオーシスを伴う神経脱落を引き起こし、マウス脳重・体重減少、寿命短縮といった症状を誘発することを明らかとした。興味深いことに、変性神経細胞では、膜タンパク質の不溶化しユビキチンやp62と共に小胞体に蓄積すること、また小胞体自体も異常に増加していることが確認された。また、クロマチン免疫沈降-DNA microarray(ChIP-chip)により、小胞体シャペロンであるGrp94等が同定され、これらが小胞体構造変化に関わっていることが示唆された。以上のことから、NF-Yが神経細胞の生存に必須であることが明らかとなると共に、その欠損が異常タンパク質の蓄積や小胞体構造異常を伴う特徴的な神経変性病態を引き起こすことが明らかとなった。

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  • ポリグルタミン病モデルを用いた神経細胞維持・変性に関わる新たな転写制御機構の同定

    研究課題/領域番号:23700430  2011年 - 2012年

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    山中 智行

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    担当区分:研究代表者  資金種別:競争的資金

    配分額:4420000円 ( 直接経費:3400000円 、 間接経費:1020000円 )

    Protein-DNAアレイ(Panomics)を用いたスクリーニングにより、ハンチントン病モデルマウスの線条体、皮質でDNA結合活性が変化している転写因子を複数同定した。このうちE-box結合因子について、変異ハンチンチン凝集体に結合すること、さらに、ChIP-chipや遺伝子発現アレイにより、その直接の下流遺伝子を同定すると共に、その一部はハンチントン病モデルマウス脳で発現低下していることも見いだされ、その病態への関与が期待された。一方、別の転写因子NF-Yについて、ノックアウトマウス解析やChIP-chip等により、NF-Yを介した遺伝子発現制御が神経変性に関わっている事も明らかとした。

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  • 選択的ニューロン病態解析法の開発・展開

    研究課題/領域番号:22110004  2010年4月 - 2015年3月

    日本学術振興会  科学研究費助成事業 新学術領域研究(研究領域提案型)  新学術領域研究(研究領域提案型)

    貫名 信行, 宮﨑 晴子, 山中 智行, 松本 弦

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    配分額:111670000円 ( 直接経費:85900000円 、 間接経費:25770000円 )

    ハンチントン病における選択的神経細胞変性の機序を明らかにするため、凝集体結合タンパク質FUS/TLS,NF-YA,p62のノックアウトマウスも含めた解析を行い、それぞれのin vivoの機能を明らかにするとともに、ポリグルタミン病病態との関連を検討した。遺伝子発現異常を呈するsodium channel beta4 subunitについて同様にノックアウトマウスの解析から線条体中型有棘神経細胞でリサージェントカレントを制御していることを示した。またその分布から大脳基底核投射線維が無髄であるという新しい解剖学的事実を提示した。またセルソーターを用いた新しい細胞機能解析手法を確立した。

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  • 転写因子NF-Yの中枢神経系における生理的・病理的役割の解明

    研究課題/領域番号:21700373  2009年 - 2010年

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    山中 智行

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    担当区分:研究代表者  資金種別:競争的資金

    配分額:4290000円 ( 直接経費:3300000円 、 間接経費:990000円 )

    転写因子NF-Yの1つの構成因子であるNF-YAについて、神経特異的コンディショナルノックアウトマウスを作製、解析したところ、出生後の体重が増加せず、離乳前に死滅してしまうことが確認された。一方、マウス脳を用いたChIP on CHIPの解析から、NF-Yターゲット候補として、遺伝子転写・翻訳やタンパク質フォールディング・分解等に関わる、様々な遺伝子が同定された。よって、NF-Yは、神経細胞において、これら遺伝子の発現を制御し、新生児期の成長に重要な役割を果たしていることが示唆された。

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  • ハンチンチン結合タンパク質のなかから同定した新規転写因子

    研究課題/領域番号:18700354  2006年 - 2007年

    日本学術振興会  科学研究費助成事業 若手研究(B)  若手研究(B)

    山中 智行

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    担当区分:研究代表者  資金種別:競争的資金

    配分額:3500000円 ( 直接経費:3500000円 )

    ハンチントン病等のポリグルタミン病は、優性遺伝性の神経変性疾患であり、原因遺伝子内の翻訳領域のCAGリピートが正常型に比べ約2〜3倍に異常に伸長することによって生じる。結果、遺伝子産物のポリグルタミン鎖が伸長され、この伸長ポリグルタミン含有タンパク質が毒性を獲得することにより、神経細胞の機能障害、変性をきたすと考えられている。病態機序としては、伸長ポリグルタミン含有タンパク質が核内に凝集体(核内封入体)を形成すること、また、核移行を人為的に阻害すると神経変性効果が劇的に減少することから、核内が主要な作用部位であると考えられている。さらに、モデルマウス等を用いた遺伝子プロファイル解析から、特定の遺伝子の発現異常が作用点の1つであると考えられている。
    私は、ハンチントン病モデル細胞から精製した核内凝集体の網羅的質量分析より、変異ハンチンチン(伸長ポリグルタミン含有ハンチンチン)に相互作用する新規タンパク質として、転写因子であるNF-Yを同定した。さらに、ハンチントン病モデルマウス脳において、変異ハンチンチンの凝集体はNF-Yを取り込むことにより、NF-Yを介したHSP70シャペロンタンパク質の転写活性を低下させることを見出した。このHSP70は変異ハンチンチンによる神経変性に抑制的に働いていることがすでに報告されている。よって、本研究より見出された変異ハンチンチンによるHSP70の発現抑制機構は、ハンチントン病の病態進行に深く関わっていることが示唆される。

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  • mPAR-6・aPKC・ASIP3者複合体の哺乳類細胞極性制御における役割の解明

    研究課題/領域番号:00J07888  2000年 - 2002年

    日本学術振興会  科学研究費助成事業 特別研究員奨励費  特別研究員奨励費

    山中 智行

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    私は、線虫胚においてaPKC、PAR-3と共に極性形成に機能するPAR-6(PDZタンパク質)の哺乳類ホモログを同定し、その上皮細胞における機能解析を行った。その結果、PAR-6がaPKC、ASIPと3者複合体を形成し、上皮細胞のタイトジャンクション付近に局在化することを見出した。また、極性化した上皮細胞のみならず、細胞間接着に伴う極性形成過程においても、上記3者複合体が細胞間接着部位へ局在化することも明らかとした。さらに、aPKC非結合型のPAR-6変異体を上皮細胞に高発現すると、細胞間接着に伴う、TJタンパク質の局在化、及びTJバリアー機能上昇の阻害が観察された。また、私は、低分子量Gタンパク質であるCdc42がその活性依存的にPAR-6を介してaPKCキナーゼ活性を制御することも明らかとした。以上のことから、PAR-6/aPKC/ASIPという新たなシグナル複合体が同定されると共に、この複合体において、PAR-6はCdc42等を介した細胞間接着のシグナルをaPKCに伝えてそれを活性化し、細胞間接着に伴うTJ形成に働いていることが示唆された。
    一方、共同研究者により、アフリカツメガエルの受精卵において、aPKC/ASIP複合体が動物極半球に非対称に局在することが明らかとされた。また、PAR-6も同様の発現パターンを示すことも報告され、カエル胚発生過程における3者複合体の機能の重要性が示唆された。これについて共同研究者と共に検討したところ、aPKC、PAR-6が、動物極半球、即ち予定外胚葉の分化に働いていることを明らかとした。さらに、aPKCのキナーゼ活性と共に、aPKC-PAR-6間の相互作用が、その機能発現に必要であることも明らかとした。以上のことから、脊椎動物の発生過程におけるaPKC/PAR-6複合体の重要性が見出されると共に、aPKCがPAR-6依存的な系路で働いている可能性が示唆された。
    以上のことから、PAR-6、aPKC、ASIPは、進化的に保存されたタンパク複合体を形成し、おそらくその一部は共通の分子メカニズムを用いて、多種多様な細胞の機能発現に関与すると考えられる。今後、この複合体を機軸として更に解析してゆくことにより、哺乳類の発生、あるいは、上皮、神経細胞等の分化した細胞においてもその細胞極性制御機構の,一端が明らかになると期待される。

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