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Kinji Ohno MD, PhDProfessor, Department of Neurogenetics and Bioinformatics, Nagoya University Graduate School of Medicine

Specialized field

Neurogenetics, Molecular mechanisms of neuromuscular transmission

Career Summary

Nagoya University School of Medicine, Japan
Internship in Internal Medicine, Nagoya National Hospital, Japan
Residency and Staff in Neurology, Nagoya National Hospital, Japan
Nagoya University Graduate School of Medicine, Japan
JSPS (Japanese Society for Promotion of Science) Research Fellow,
Department of Biomedical Chemistry, Nagoya University School of Medicine, Japan
Research Fellow, Department of Neurology, Mayo Clinic, USA
Research Associate, Department of Neurology, Mayo Clinic, USA
Assistant Professor, Mayo Medical School, USA
Professor, Division of Neurogenetics, Center for Neurological Diseases and Cancer, Nagoya University Graduate School of Medicine, Japan

Research Theme

Molecular pathomechanisms and the regulations of defective neuromuscular transmission defects

Research Summary

A. Project AIM
Acetylcholinesterase (AChE) at the neuromuscular junction (NMJ) is anchored to the synaptic basal lamina via a triple helical collagen Q (ColQ). Congenital defect of ColQ causes endplate AChE deficiency. We previously report that the recombinant human ColQ-tailed AChE can specifically anchor to the frog NMJ in vitro. This observation prompted us to hypothesize that ColQ has a proprietary targeting signal for the synaptic basal lamina and that ColQ-tailed AChE expressed in a limited number of skeletal muscles is efficiently propagated and anchored to the NMJs. We tested this hypothesis in Colq-/- mice to develop a rationale therapy for endplate AChE deficiency, as well as for congenital defects of other extracellular matrix proteins.

B. Achievements
We constructed a recombinant adeno-associated virus serotype 8 (AAV8) carrying human COLQ. We injected AAV8-COLQ to the tail vein of Colq-/- mice. In four weeks after injection, the NMJ ultrastructure was normalized, and electrophysiological features of the NMJ signal transmission were normalized but not completely. In six weeks after injection, motor functions of the treated mice normalized completely; ColQ-tailed AChE was specifically anchored to the NMJ by immunostaining; the amount of AChE was restored to 89% of the wild-type. We also injected AAV8-COLQ intramuscularly to the right anterior tibial muscle, and found that ColQ-tailed AChE was expressed to ~15% of the wild-type in non-injected forelimbs and left hindlimb. We next characterized the molecular basis of this efficient recovery. We first confirmed that ColQ-tailed AChE can be specifically targeted to NMJ by an in vitro overlay assay in Colq-/- mice muscle sections. We then injected AAV1-COLQ-IRES-EGFP into the left tibialis anterior and detected AChE in non-injected limbs. Furthermore, in vivo injection of recombinant ColQ-tailed AChE protein complex into the gluteus maximus muscle of Colq-/- mice led to accumulation of AChE in non-injected forelimbs. We demonstrated for the first time in vivo that the ColQ protein contains a tissue-targeting signal that is sufficient for anchoring itself to the NMJ. We propose that the protein-anchoring therapy is potentially applicable to a broad spectrum of diseases affecting extracellular matrix molecules.

Principal Research Achievement

  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, Ito M, 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. 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)
  22. 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 patiens. Neurology, 73, 228-235 (2009)
  23. Itoh T, Fujita Y, Ito M, Masuda A, Ohno K, Ichihara M, Kojima T, Nozawa Y, Ito M. Molecular hydrogen suppresses FcepsilonRI-mediated signal transduction and prevents degranulation of mast cells. Biochem Biophys Res Commun, 389, 651-656 (2009)
  24. Gao K, Masuda A, Matsuura T, Ohno K. Human branch point consensus sequence is yUnAy. Nucleic Acids Res, 36, 2257-2267 (2008)
  25. Ito M, Masuda A, Jinno S, Katagiri T, Krejci E, Ohno K. Viral vector-medicated expression of human collagen Q in cultured cells. Chem Biol Interact, 175, 346-348 (2008)
  26. Kurosaki T, Matsuura T, Ohno K, Ueda S. Long-range PCR for the diagnosis of spinocerebellar ataxia type 10. Neurogenetics, 9, 151-152 (2008)
  27. 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)
  28. 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)
  29. Shen XM, Fukuda T, Ohno K, Sine SM, Engel AG. Congenital myasthenia-related AChR delta subunit mutation interferes with intersubunit communication essential for channel gating. J Clin Invest, 118, 1867-1876 (2008)
  30. Ichihara M et al. Thermodynamic instability of siRNA duplex is a prerequisite for dependable prediction of siRNA activities. Nucleic Acids Res. 35:e123 (2007)
  31. Masuda A et al. Essential role of GATA transcriptional factors in the activation of mast cells. J Immunol 178:360-368 (2007)
  32. 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)
  33. 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)
  34. Cai Y et al. Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders. EMBO J. 23: 2047-2058 (2004)
  35. Ohno K et al. A frameshifting mutation in CHRNE unmasks skipping of the preceding exon. Hum. Mol. Genet. 12: 3055-3066 (2003)
  36. 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)
  37. Tsujino A et al. Myasthenic syndrome caused by mutation of the SCN4A sodium channel. Proc. Natl. Acad. Sci. USA. 100: 7377-7382 (2003)
  38. 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)
  39. Engel AG et al. Neurological diseases: Sleuthing molecular targets for neurological diseases at the neuromuscular junction. Nat. Rev. Neurosci. 4: 339-352 (2003)
  40. Ohno K et al. Rapsyn mutations in humans cause endplate acetylcholine-receptor deficiency and myasthenic syndrome. Am. J. Hum. Genet. 70: 875-885 (2002)
  41. 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)
  42. 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)
  43. Wang H-L et al. Acetylcholine receptor M3 domain: stereochemical and volume contributions to channel gating. Nat. Neurosci. 2: 226-233 (1999)
  44. 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)
  45. 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)
  46. 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)
  47. 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)
  48. 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)
  49. 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)


1995 Neurology Research Award, Mayo Clinic