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大野 欽司 Kinji Ohno 名古屋大学大学院医学系研究科神経遺伝情報学・教授・医学博士


専門分野

神経遺伝情報学・神経筋接合部分子病態機構

略歴

1983年3月
名古屋大学医学部医学科卒業
1983年6月
国立名古屋病院研修医
1985年4月
国立名古屋病院神経内科レジデント及びスタッフ
1988年4月
名古屋大学大学院医学系研究科(神経内科学専攻)
1992年4月
日本学術振興会特別研究員(名古屋大学医学部第2生化学)
1993年4月
Research Fellow, Dept of Neurology, Mayo Clinic
1996年4月
Research Associate, Dept of Neurology, Mayo Clinic
1998年4月
Assistant Professor, Mayo Medical School
2004年9月
名古屋大学大学院医学系研究科 神経遺伝情報学 教授

研究課題名

神経筋接合部分子欠損症の分子病態とその制御

これまでの成果と今後の展望

A. 研究目的
人工的な遺伝子導入による遺伝子治療は各種分子欠損モデル細胞において顕著な効果を示す。一方、モデル動物ならびにヒトに対する応用は、導入遺伝子の時空間的な発現調節が困難であることに加えて、組織特異的な遺伝子導入が困難であるために、細胞治療に比して困難が多い。我々は先天性筋無力症候群の一型の終板アセチルコリンエステラーゼ(acetylcholinesterase, AChE)欠損の研究の過程で、患者において欠損をする細胞外分子collagen Q (ColQ)分子自身が組織標的シグナルを持ちperlecan, MuSKに結合をすることを同定した。ColQが組織標的シグナルを持つ細胞外分子であることを利用して、タンパク自身に標的組織へのanchoringを行わせるprotein-anchoring therapyを提唱し、本手法の有用性をColqノックアウトによるAChE欠損モデルマウスを用いて検証を行った。

B. 研究成果
ヒトColQ分子のマウス神経筋接合部へのin vitro anchoringを検証した後、ヒトCOLQ遺伝子を組み込んだAAV8 (adeno-associated virus serotype 8)を作成し、5週齢のColqノックアウトマウスの尾静脈より注射を行ったところマウスの運動機能をほぼ正常化できた。易疲労性試験でも正常マウスと同様に易疲労性が消失した。さらに、骨格筋切片でもcolQとAChEの神経筋接合部への係留を確認した。ショ糖濃度勾配遠心にてAChE-ColQ複合体の分画を行ったところ、Colqノックアウトマウスには存在しないAChE-ColQ複合体がAAV8-COLQ治療により正常マウス骨格筋と同様のAChE-ColQ複合体分画が得られ、AChE-ColQ複合体量は正常マウスの89%のレベルまで改善した。電顕による観察でも多くの神経筋接合部の超微構造が改善をしていた。さらに電気生理学的検討でもMEPP, EPPの減衰時間・振幅ともに正常に近づいた。定量PCRによる解析では約30%の骨格筋がAAV8-COLQに感染をしており、タンパク量が正常の89%まで改善したことは、感染をした骨格筋より放出された非対称性AChEが周囲のシナプス基底膜に運ばれたと推察された。いずれのアッセイ手法を用いても予想以上に良好な効果が得られておりprotein anchoring therapyの有用性が示唆された。さらに血液移行が少ないAAV1-COLQの下肢筋注、ならびに精製AChE-ColQ複合体タンパクの下肢筋注にて、上肢神経筋接合部におけるAChE, ColQの発現を認めた。一方、コントロールとして用いたAAV1-EGFPは注射部位近傍の骨格筋のみに発現をしており、protein-anchoring therapyの有用性が示めされた。

主要研究業績

  1. Ito M, Suzuki Y, Okada T, Fukudome T, Yoshimura T, Masuda A, Takeda S, Krejci E, Ohno K: Protein-anchoring strategy for delivering acetylcholinesterase to the neuromuscular junction. Mol Ther, in press (2012)
  2. Masuda A, Andersen HS, Doktor TK, Okamoto T, Ito M, Andresen BS, Ohno K: Cugbp1 and mbnl1 preferentially bind to 3' utrs and facilitate mrna decay. Sci Rep, 2, 209 (2012)
  3. Matsuura T, Minami N, Arahata H, Ohno K, Abe K, Hayashi YK, Nishino I: Myotonic dystrophy type 2 is rare in the japanese population. J Hum Genet, 57, 219-220 (2012)
  4. Ohno K, Ito M, Engel A. Congenital myasthenic syndromes ? molecular bases of congenital defects of proteins at the neuromuscular junction ?. In Myopathy, (Rijeka, InTech), p. in press (2012)
  5. Ohno K, Ito M, Ichihara M, Ito M. Molecular hydrogen as an emerging therapeutic medical gas for neurodegenerative and other diseases. In Oxidative medicine and cellular longevity, Pereira M, ed. (New York, Hindawi Publishing Corp.), p. in press (2012)
  6. Yamashita Y, Matsuura T, Shinmi J, Amakusa Y, Masuda A, Ito M, Kinoshita M, Furuya H, Abe K, Ibi T, Sahashi K, Ohno K: Four parameters increase the sensitivity and specificity of the exon array analysis and disclose twenty-five novel aberrantly spliced exons in myotonic dystrophy. J Hum Genet, in press (2012)
  7. Yoshinaga H, Sakoda S, Good JM, Takahashi MP, Kubota T, Arikawa-Hirasawa E, Nakata T, Ohno K, Kitamura T, Kobayashi K, Ohtsuka Y: A novel mutation in scn4a causes severe myotonia and school-age-onset paralytic episodes. J Neurol Sci, 315, 15-19 (2012)
  8. Fu Y, Masuda A, Ito M, Shinmi J, Ohno K: Ag-dependent 3'-splice sites are predisposed to aberrant splicing due to a mutation at the first nucleotide of an exon. Nucleic Acids Res, 39, 4396-4404 (2011)
  9. Hirayama M, Nakamura T, Watanabe H, Uchida K, Hama T, Hara T, Niimi Y, Ito M, Ohno K, Sobue G: Urinary 8-hydroxydeoxyguanosine correlate with hallucinations rather than motor symptoms in parkinson's disease. Parkinsonism Relat Disord, 17, 46-49 (2011)
  10. Ito M, Ibi T, Sahashi K, Ichihara M, Ohno K: Open-label trial and randomized, double-blind, placebo-controlled, crossover trial of hydrogen-enriched water for mitochondrial and inflammatory myopathies. Med Gas Res, 1, 24 (2011)
  11. Itoh T, Hamada N, Terazawa R, Ito M, Ohno K, Ichihara M, Nozawa Y: Molecular hydrogen inhibits lipopolysaccharide/interferon gamma-induced nitric oxide production through modulation of signal transduction in macrophages. Biochem Biophys Res Commun, 411, 143-149 (2011)
  12. Kaneko H, Kitoh H, Matsuura T, Masuda A, Ito M, Mottes M, Rauch F, Ishiguro N, Ohno K: Hyperuricemia cosegregating with osteogenesis imperfecta is associated with a mutation in gpatch8. Hum Genet, 130, 671-683 (2011)
  13. Kawakami Y, Ito M, Hirayama M, Sahashi K, Ohkawara B, Masuda A, Nishida H, Mabuchi N, Engel AG, Ohno K: Anti-musk autoantibodies block binding of collagen q to musk. Neurology, 77, 1819-1826 (2011)
  14. Ohno K: Genetic defects and disorders at the neuromuscular junction. Brain Nerve, 63, 669-678 (2011)
  15. Ohno K, Engel A. Molecular defects of acetylcholine receptor subunits in congenital myasthenic syndromes. In Pharmacology of nicotinic acetylcholine receptors from the basic and therapeutic perspectives, Arias HR, ed. (Kerala, Research Signpost), pp. 175-186 (2011)
  16. Ohno K, Masuda A. Rna pathologies in neurological disorders. In Neurochemical mechanisms in disease, advances in neurobiology, Lajtha A, ed. (New York, Springer), pp. 399-415 (2011)
  17. Selcen D, Juel VC, Hobson-Webb LD, Smith EC, Stickler DE, Bite AV, Ohno K, Engel AG: Myasthenic syndrome caused by plectinopathy. Neurology, 76, 327-336 (2011)
  18. Almeida T, Alonso I, Martins S, Ramos EM, Azevedo L, Ohno K, Amorim A, Saraiva-Pereira ML, Jardim LB, Matsuura T, Sequeiros J, Silveira I: Ancestral origin of the attct repeat expansion in spinocerebellar ataxia type 10 (sca10). PLoS One, 4, e4553 (2009)
  19. Bian Y, Masuda A, Matsuura T, Ito M, Okushin K, Engel AG, Ohno K: Tannic acid facilitates expression of the polypyrimidine tract binding protein and alleviates deleterious inclusion of chrna1 exon p3a due to an hnrnp h-disrupting mutation in congenital myasthenic syndrome. Hum Mol Genet, 18, 1229-1237 (2009)
  20. Fu Y, Ito M, Fujita Y, Ichihara M, Masuda A, Suzuki Y, Maesawa S, Kajita Y, Hirayama M, Ohsawa I, Ohta S, Ohno K: Molecular hydrogen is protective against 6-hydroxydopamine-induced nigrostriatal degeneration in a rat model of parkinson's disease. Neurosci Lett, 453, 81-85 (2009)
  21. Itoh T, Fujita Y, Ito M, Masuda A, Ohno K, Ichihara M, Kojima T, Nozawa Y: Molecular hydrogen suppresses fcepsilonri-mediated signal transduction and prevents degranulation of mast cells. Biochem Biophys Res Commun, 389, 651-656 (2009)
  22. Kurosaki T, Matsuura T, Ohno K, Ueda S: Alu-mediated acquisition of unstable attct pentanucleotide repeats in the human atxn10 gene. Mol Biol Evol, 26, 2573-2579 (2009)
  23. Milone M, Shen XM, Selcen D, Ohno K, Brengman J, Iannaccone ST, Harper CM, Engel AG: Myasthenic syndrome due to defects in rapsyn: Clinical and molecular findings in 39 patients. Neurology, 73, 228-235 (2009)
  24. Ohno K: Aberrant pre-mrna splicing in neurological disorders. Tanpakushitsu Kakusan Koso, 54, 2239-2244 (2009)
  25. Ohno K, Ito M, Masuda A: Molecular bases and therapeutic strategies in defective neuromuscular transmissions: Lessons learned from a prototypical synapse. Nihon Shinkei Seishin Yakurigaku Zasshi, 29, 145-151 (2009)
  26. Gao K, Masuda A, Matsuura T, Ohno K: Human branch point consensus sequence is yunay. Nucleic Acids Res, 36, 2257-2267 (2008)
  27. Ito M, Masuda A, Jinno S, Katagiri T, Krejci E, Ohno K: Viral vector-mediated [corrected] expression of human collagen q in cultured cells. Chem Biol Interact, 175, 346-348 (2008)
  28. Kurosaki T, Matsuura T, Ohno K, Ueda S: Long-range pcr for the diagnosis of spinocerebellar ataxia type 10. Neurogenetics, 9, 151-152 (2008)
  29. Masuda A, Shen XM, Ito M, Matsuura T, Engel AG, Ohno K: Hnrnp h enhances skipping of a nonfunctional exon p3a in chrna1 and a mutation disrupting its binding causes congenital myasthenic syndrome. Hum Mol Genet, 17, 4022-4035 (2008)
  30. Saito T, Amakusa Y, Kimura T, Yahara O, Aizawa H, Ikeda Y, Day JW, Ranum LP, Ohno K, Matsuura T: Myotonic dystrophy type 2 in japan: Ancestral origin distinct from caucasian families. Neurogenetics, 9, 61-63 (2008)
  31. Shen X-M et al. Congenital myasthenia-related AChR delta subunit mutation interferes with intersubunit communication essential for channel gating. J. Clin. Invest. 118: 1867-1876 (2008)
  32. Gao K et al. Human branch point consensus sequence is yUnAy. Nucleic Acids Res. 36: 2257-2267 (2008)
  33. Ichihara M et al. Thermodynamic instability of siRNA duplex is a prerequisite for dependable prediction of siRNA activities. Nucleic Acids Res. 35:e123 (2007)
  34. Masuda A et al. Essential role of GATA transcriptional factors in the activation of mast cells. J Immunol 178:360-368 (2007)
  35. Sahashi K et al. In vitro and in silico analysis reveals an efficient algorithm to predict the splicing consequences of mutations at the 5' splice sites. Nucleic Acids Res. 35: 5995-6003 (2007)
  36. Shen X-M et al. Subunit-specific contribution to agonist binding and channel gating revealed by inherited mutation in muscle acetylcholine receptor M3-M4 linker. Brain 128: 345-355 (2005)
  37. Cai Y et al. Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders. EMBO J. 23: 2047-2058 (2004)
  38. Ohno K et al. A frameshifting mutation in CHRNE unmasks skipping of the preceding exon. Hum. Mol. Genet. 12: 3055-3066 (2003)
  39. Ohno K. et al. E-box mutations in the RAPSN promoter region in eight cases with congenital myasthenic syndrome. Hum. Mol. Genet. 12: 739-748 (2003)
  40. Tsujino A et al. Myasthenic syndrome caused by mutation of the SCN4A sodium channel. Proc. Natl. Acad. Sci. USA. 100: 7377-7382 (2003)
  41. Shen X-M et al. Mutation causing severe myasthenia reveals functional asymmetry of AChR signature cystine loops in agonist binding and gating. J. Clin. Invest. 111: 497-505 (2003)
  42. Engel AG et al. Neurological diseases: Sleuthing molecular targets for neurological diseases at the neuromuscular junction. Nat. Rev. Neurosci. 4: 339-352 (2003)
  43. Ohno K et al. Rapsyn mutations in humans cause endplate acetylcholine-receptor deficiency and myasthenic syndrome. Am. J. Hum. Genet. 70: 875-885 (2002)
  44. Ohno K. et al. Choline acetyltransferase mutations cause myasthenic syndrome associated with episodic apnea in humans. Proc. Natl. Acad. Sci. USA. 98: 2017-2022 (2001)
  45. Ohno K. et al. Congenital end-plate acetylcholinesterase deficiency caused by a nonsense mutation and an A-->G splice-donor-site mutation at position +3 of the collagenlike-tail-subunit gene (COLQ): how does G at position +3 result in aberrant splicing? Am. J. Hum. Genet. 65: 635-644 (1999)
  46. Wang H-L et al. Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating. Nat. Neurosci. 2: 226-233 (1999)
  47. Ohno K. et al. Human endplate acetylcholinesterase deficiency caused by mutations in the collagen-like tail subunit (ColQ) of the asymmetric enzyme. Proc. Natl. Acad. Sci. USA. 95: 9654-9659 (1998)
  48. Milone M et al. Mode switching kinetics produced by a naturally occurring mutation in the cytoplasmic loop of the human acetylcholine receptor epsilon subunit. Neuron 20: 575-588 (1998)
  49. Milone M et al. Slow-channel myasthenic syndrome caused by enhanced activation, desensitization, and agonist binding affinity attributable to mutation in the M2 domain of the acetylcholine receptor alpha subunit. J. Neurosci. 17: 5651-5665 (1997)
  50. Ohno K et al. Congenital myasthenic syndrome caused by decreased agonist binding affinity due to a mutation in the acetylcholine receptor epsilon subunit. Neuron 17: 157-170 (1996)
  51. Ohno K. Congenital myasthenic syndrome caused by prolonged acetylcholine receptor channel openings due to a mutation in the M2 domain of the epsilon subunit. Proc. Natl. Acad. Sci. USA. 92: 758-762 (1995)
  52. Sine SM et al. Mutation of the acetylcholine receptor alpha subunit causes a slow-channel myasthenic syndrome by enhancing agonist binding affinity. Neuron 15: 229-239 (1995)

受賞

1995 年 Neurology Research Award, Mayo Clinic

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