Kazuhiro Kobayashi

Publications (34)192.46 Total impact

• Article: Impaired viability of muscle precursor cells in muscular dystrophy with glycosylation defects and amelioration of its severe phenotype by limited gene expression.

  Motoi Kanagawa, Chih-Chieh Yu, Chiyomi Ito, So-Ichiro Fukada, Masako Hozoji-Inada, Tomoko Chiyo, Atsushi Kuga, Megumi Matsuo, Kanoko Sato, Masahiko Yamaguchi, Takahito Ito, Yoshihisa Ohtsuka, Yuki Katanosaka, Yuko Miyagoe-Suzuki, Keiji Naruse, Kazuhiro Kobayashi, Takashi Okada, Shin'ichi Takeda, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: A group of muscular dystrophies, dystroglycanopathy, is caused by abnormalities in post-translational modifications of dystroglycan (DG). To better understand the pathophysiological roles of DG modification and to establish effective clinical treatment for dystroglycanopathy, we here generated 2 distinct conditional knock-out (cKO) mice for fukutin, the first dystroglycanopathy gene identified for Fukuyama congenital muscular dystrophy. The first dystroglycanopathy model-myofiber-selective fukutin-cKO (MCK-fukutin-cKO) mice-showed mild muscular dystrophy. Forced exercise experiments in presymptomatic MCK-fukutin-cKO mice revealed that myofiber membrane fragility triggered disease manifestation. The second dystroglycanopathy model-muscle precursor cell (MPC)-selective cKO (Myf5-fukutin-cKO) mice-exhibited more severe phenotypes of muscular dystrophy. Using an isolated MPC culture system, we demonstrated, for the first time, that defects in the fukutin-dependent modification of DG lead to impairment of MPC proliferation, differentiation, and muscle regeneration. These results suggest that impaired MPC viability contributes to the pathology of dystroglycanopathy. Since our data suggested that frequent cycles of myofiber degeneration/regeneration accelerate substantial and/or functional loss of MPC, we expected that protection from disease-triggering myofiber degeneration provides therapeutic effects even in mouse models with MPC defects; therefore, we restored fukutin expression in myofibers. Adeno-associated virus (AAV)-mediated rescue of fukutin expression that was limited in myofibers successfully ameliorated the severe pathology even after disease progression. In addition, compared to other gene therapy studies, considerably low AAV titers were associated with therapeutic effects. Together, our findings indicated that fukutin-deficient dystroglycanopathy is a regeneration-defective disorder, and gene therapy is a feasible treatment for the wide range of dystroglycanopathy even after disease progression.
  Human Molecular Genetics 04/2013; · 7.64 Impact Factor
• Article: Detection of the dystroglycanopathy protein, fukutin, using a new panel of site-specific monoclonal antibodies.

  Tracy A Lynch, Le Thanh Lam, Nguyen thi Man, Kazuhiro Kobayashi, Tatsushi Toda, Glenn E Morris
  [show abstract] [hide abstract]
  ABSTRACT: Mutations in the gene encoding fukutin protein cause Fukuyama muscular dystrophy, a severe congenital disorder that occurs mainly in Japan. A major consequence of the mutation is reduced glycosylation of alpha-dystroglycan, which is also a feature of other forms of congenital and limb-girdle muscular dystrophy. Immunodetection of endogenous fukutin in cells and tissues has been difficult and this has hampered progress in understanding fukutin function and disease pathogenesis. Using a new panel of monoclonal antibodies which bind to different defined sites on the fukutin molecule, we now show that fukutin has the predicted size for a protein without extensive glycosylation and is present at the Golgi apparatus at very low levels. These antibodies should enable more rapid future progress in understanding the molecular function of fukutin.
  Biochemical and Biophysical Research Communications 07/2012; 424(2):354-7. · 2.48 Impact Factor
• Article: Age and origin of the FCMD 3′-untranslated-region retrotransposal insertion mutation causing Fukuyama-type congenital muscular dystrophy in the Japanese population

  Roberto Colombo, Angelo A. Bignamini, Anna Carobene, Junko Sasaki, Masashi Tachikawa, Kazuhiro Kobayashi, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: Fukuyama-type congenital muscular dystrophy (FCMD), an autosomal recessive disorder with a high prevalence in the Japanese population, is characterised by severe muscular dystrophy associated with brain malformation (cortical dysgenesis) and mental retardation. In Japan, 87% of FCMD-bearing chromosomes carry a 3-kb retrotransposal insertion of tandemly repeated sequences within the disease gene recently identified on chromosome 9q31, and most of them share a common founder haplotype. FCMD is the first human disease known to be caused primarily by an ancient retrotransposal integration. By applying two methods for the study of linkage disequilibrium between flanking polymorphic markers and the disease locus, and of its decay over time, the age of the insertion mutation causing FCMD in Japanese patients is calculated to be approximately 102 generations (95% confidence interval: 86–117 g), or slightly less. The estimated age dates the most recent common ancestor of the mutation-bearing chromosomes back to the time (or a few centuries before) the Yayoi people started migrating to Japan from the Korean peninsula. This finding makes the molecular population genetics of FCMD understandable in the context of Japan’s history and the founder effect consistent with the prevalent theory on the origins of the modern Japanese population.
  Human Genetics 04/2012; 107(6):559-567. · 5.07 Impact Factor
• Article: Mislocalization of fukutin protein by disease-causing missense mutations can be rescued with treatments directed at folding amelioration.

  Masaji Tachikawa, Motoi Kanagawa, Chih-Chieh Yu, Kazuhiro Kobayashi, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: Fukuyama-type congenital muscular dystrophy (FCMD), the second most common childhood muscular dystrophy in Japan, is caused by alterations in the fukutin gene. Mutations in fukutin cause abnormal glycosylation of α-dystroglycan, a cell surface laminin receptor; however, the exact function and pathophysiological role of fukutin are unclear. Although the most prevalent mutation in Japan is a founder retrotransposal insertion, point mutations leading to abnormal glycosylation of α-dystroglycan have been reported, both in Japan and elsewhere. To understand better the molecular pathogenesis of fukutin-deficient muscular dystrophies, we constructed 13 disease-causing missense fukutin mutations and examined their pathological impact on cellular localization and α-dystroglycan glycosylation. When expressed in C2C12 myoblast cells, wild-type fukutin localizes to the Golgi apparatus, whereas the missense mutants A170E, H172R, H186R, and Y371C instead accumulated in the endoplasmic reticulum. Protein O-mannose β1,2-N-acetylglucosaminyltransferase 1 (POMGnT1) also mislocalizes when co-expressed with these missense mutants. The results of nocodazole and brefeldin A experiments suggested that these mutant proteins were not transported to the Golgi via the anterograde pathway. Furthermore, we found that low temperature culture or curcumin treatment corrected the subcellular location of these missense mutants. Expression studies using fukutin-null mouse embryonic stem cells showed that the activity responsible for generating the laminin-binding glycan of α-dystroglycan was retained in these mutants. Together, our results suggest that some disease-causing missense mutations cause abnormal folding and localization of fukutin protein, and therefore we propose that folding amelioration directed at correcting the cellular localization may provide a therapeutic benefit to glycosylation-deficient muscular dystrophies.
  Journal of Biological Chemistry 01/2012; 287(11):8398-406. · 4.77 Impact Factor
• Article: Absence of post-phosphoryl modification in dystroglycanopathy mouse models and wild-type tissues expressing non-laminin binding form of α-dystroglycan.

  Atsushi Kuga, Motoi Kanagawa, Atsushi Sudo, Yiumo Michael Chan, Michiko Tajiri, Hiroshi Manya, Yamato Kikkawa, Motoyoshi Nomizu, Kazuhiro Kobayashi, Tamao Endo, Qi L Lu, Yoshinao Wada, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: α-Dystroglycan (α-DG) is a membrane-associated glycoprotein that interacts with several extracellular matrix proteins, including laminin and agrin. Aberrant glycosylation of α-DG disrupts its interaction with ligands and causes a certain type of muscular dystrophy commonly referred to as dystroglycanopathy. It has been reported that a unique O-mannosyl tetrasaccharide (Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,2-Man) and a phosphodiester-linked modification on O-mannose play important roles in the laminin binding activity of α-DG. In this study, we use several dystroglycanopathy mouse models to demonstrate that, in addition to fukutin and LARGE, FKRP (fukutin-related protein) is also involved in the post-phosphoryl modification of O-mannose on α-DG. Furthermore, we have found that the glycosylation status of α-DG in lung and testis is minimally affected by defects in fukutin, LARGE, or FKRP. α-DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results show that FKRP is involved in post-phosphoryl modification rather than in O-mannosyl tetrasaccharide synthesis. Our data also demonstrate that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of α-DG functional expression as a laminin receptor in normal tissues and cells.
  Journal of Biological Chemistry 01/2012; 287(12):9560-7. · 4.77 Impact Factor
• Article: Absence of post-phosphoryl modification in dystroglycanopathy mouse models and wild-type tissues expressing a non-laminin binding form of alpha-dystroglycan

  Atsushi Kuga, Motoi Kanagawa, Atsushi Sudo, Yiumo Michael Chan, Michiko Tajiri, Hiroshi Manya, Yamato Kikkawa, Motoyoshi Nomizu, Kazuhiro Kobayashi, Tamao Endo, Qi L. Lu, Yoshinao Wada, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: Alpha-dystroglycan (α-DG) is a membrane-associated glycoprotein that interacts with several extracellular matrix proteins, including laminin and agrin. Aberrant glycosylation of α-DG disrupts its interaction with ligands and causes a certain type of muscular dystrophy commonly referred to as dystroglycanopathy. It has been reported that a unique O-mannosyl tetrasaccharide (Neu5Ac-α2,3-Gal-β1,4-GlcNAc-β1,2-Man) and a phosphodiester-linked modification on O-mannose play important roles in the laminin binding activity of α-DG. In this study, we use several dystroglycanopathy mouse models to demonstrate that, in addition to fukutin and LARGE, fukutin-related protein (FKRP) is also involved in the post-phosphoryl modification of O-mannose on α-DG. Furthermore, we have found that the glycosylation status of α-DG in lung and testis is minimally affected by defects in fukutin, LARGE or FKRP. α-DG prepared from wild-type lung- or testis-derived cells lacks the post-phosphoryl moiety and shows little laminin-binding activity. These results show that FKRP is involved in post-phosphoryl modification rather than in O-mannosyl tetrasaccharide synthesis. Our data also demonstrate that post-phosphoryl modification not only plays critical roles in the pathogenesis of dystroglycanopathy but also is a key determinant of α-DG functional expression as a laminin receptor in normal tissues and cells.
  Journal of Biological Chemistry 01/2012; · 4.77 Impact Factor
• Article: Molecular targeting therapy for fukuyama muscular dystrophy.

  Tatsushi Toda, Mariko Taniguchi-Ikeda, Kazuhiro Kobayashi
  Rinshō shinkeigaku = Clinical neurology. 01/2012; 52(11):1158.
• Source

  Available from: Hasan Orhan

  Article: Pathogenic exon-trapping by SVA retrotransposon and rescue in Fukuyama muscular dystrophy.

  Mariko Taniguchi-Ikeda, Kazuhiro Kobayashi, Motoi Kanagawa, Chih-chieh Yu, Kouhei Mori, Tetsuya Oda, Atsushi Kuga, Hiroki Kurahashi, Hasan O Akman, Salvatore DiMauro, Ryuji Kaji, Toshifumi Yokota, Shin'ichi Takeda, Tatsushi Toda
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  ABSTRACT: Fukuyama muscular dystrophy (FCMD; MIM253800), one of the most common autosomal recessive disorders in Japan, was the first human disease found to result from ancestral insertion of a SINE-VNTR-Alu (SVA) retrotransposon into a causative gene. In FCMD, the SVA insertion occurs in the 3' untranslated region (UTR) of the fukutin gene. The pathogenic mechanism for FCMD is unknown, and no effective clinical treatments exist. Here we show that aberrant messenger RNA (mRNA) splicing, induced by SVA exon-trapping, underlies the molecular pathogenesis of FCMD. Quantitative mRNA analysis pinpointed a region that was missing from transcripts in patients with FCMD. This region spans part of the 3' end of the fukutin coding region, a proximal part of the 3' UTR and the SVA insertion. Correspondingly, fukutin mRNA transcripts in patients with FCMD and SVA knock-in model mice were shorter than the expected length. Sequence analysis revealed an abnormal splicing event, provoked by a strong acceptor site in SVA and a rare alternative donor site in fukutin exon 10. The resulting product truncates the fukutin carboxy (C) terminus and adds 129 amino acids encoded by the SVA. Introduction of antisense oligonucleotides (AONs) targeting the splice acceptor, the predicted exonic splicing enhancer and the intronic splicing enhancer prevented pathogenic exon-trapping by SVA in cells of patients with FCMD and model mice, rescuing normal fukutin mRNA expression and protein production. AON treatment also restored fukutin functions, including O-glycosylation of α-dystroglycan (α-DG) and laminin binding by α-DG. Moreover, we observe exon-trapping in other SVA insertions associated with disease (hypercholesterolemia, neutral lipid storage disease) and human-specific SVA insertion in a novel gene. Thus, although splicing into SVA is known, we have discovered in human disease a role for SVA-mediated exon-trapping and demonstrated the promise of splicing modulation therapy as the first radical clinical treatment for FCMD and other SVA-mediated diseases.
  Nature 10/2011; 478(7367):127-31. · 36.28 Impact Factor
• Article: Post-translational maturation of dystroglycan is necessary for pikachurin binding and ribbon synaptic localization.

  Motoi Kanagawa, Yoshihiro Omori, Shigeru Sato, Kazuhiro Kobayashi, Yuko Miyagoe-Suzuki, Shin'ichi Takeda, Tamao Endo, Takahisa Furukawa, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: Pikachurin, the most recently identified ligand of dystroglycan, plays a crucial role in the formation of the photoreceptor ribbon synapse. It is known that glycosylation of dystroglycan is necessary for its ligand binding activity, and hypoglycosylation is associated with a group of muscular dystrophies that often involve eye abnormalities. Because little is known about the interaction between pikachurin and dystroglycan and its impact on molecular pathogenesis, here we characterize the interaction using deletion constructs and mouse models of muscular dystrophies with glycosylation defects (Large(myd) and POMGnT1-deficient mice). Pikachurin-dystroglycan binding is calcium-dependent and relatively less sensitive to inhibition by heparin and high NaCl concentration, as compared with other dystroglycan ligand proteins. Using deletion constructs of the laminin globular domains in the pikachurin C terminus, we show that a certain steric structure formed by the second and the third laminin globular domains is necessary for the pikachurin-dystroglycan interaction. Binding assays using dystroglycan deletion constructs and tissue samples from Large-deficient (Large(myd)) mice show that Large-dependent modification of dystroglycan is necessary for pikachurin binding. In addition, the ability of pikachurin to bind to dystroglycan prepared from POMGnT1-deficient mice is severely reduced, suggesting that modification of the GlcNAc-β1,2-branch on O-mannose is also necessary for the interaction. Immunofluorescence analysis reveals a disruption of pikachurin localization in the photoreceptor ribbon synapse of these model animals. Together, our data demonstrate that post-translational modification on O-mannose, which is mediated by Large and POMGnT1, is essential for pikachurin binding and proper localization, and suggest that their disruption underlies the molecular pathogenesis of eye abnormalities in a group of muscular dystrophies.
  Journal of Biological Chemistry 10/2010; 285(41):31208-16. · 4.77 Impact Factor
• Article: Post-translational Maturation of Dystroglycan Is Necessary for Pikachurin Binding and Ribbon Synaptic Localization

  Motoi Kanagawa, Yoshihiro Omori, Shigeru Sato, Kazuhiro Kobayashi, Yuko Miyagoe-Suzuki, Shin'ichi Takeda, Tamao Endo, Takahisa Furukawa, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: Pikachurin, the most recently identified ligand of dystroglycan, plays a crucial role in the formation of the photoreceptor ribbon synapse. It is known that glycosylation of dystroglycan is necessary for its ligand binding activity, and hypoglycosylation is associated with a group of muscular dystrophies that often involve eye abnormalities. Because little is known about the interaction between pikachurin and dystroglycan and its impact on molecular pathogenesis, here we characterize the interaction using deletion constructs and mouse models of muscular dystrophies with glycosylation defects (Largemyd and POMGnT1-deficient mice). Pikachurin-dystroglycan binding is calcium-dependent and relatively less sensitive to inhibition by heparin and high NaCl concentration, as compared with other dystroglycan ligand proteins. Using deletion constructs of the laminin globular domains in the pikachurin C terminus, we show that a certain steric structure formed by the second and the third laminin globular domains is necessary for the pikachurin-dystroglycan interaction. Binding assays using dystroglycan deletion constructs and tissue samples from Large-deficient (Largemyd) mice show that Large-dependent modification of dystroglycan is necessary for pikachurin binding. In addition, the ability of pikachurin to bind to dystroglycan prepared from POMGnT1-deficient mice is severely reduced, suggesting that modification of the GlcNAc-β1,2-branch on O-mannose is also necessary for the interaction. Immunofluorescence analysis reveals a disruption of pikachurin localization in the photoreceptor ribbon synapse of these model animals. Together, our data demonstrate that post-translational modification on O-mannose, which is mediated by Large and POMGnT1, is essential for pikachurin binding and proper localization, and suggest that their disruption underlies the molecular pathogenesis of eye abnormalities in a group of muscular dystrophies.
  Journal of Biological Chemistry 10/2010; 285(41):31208-31216. · 4.77 Impact Factor
• Article: Fukutin gene retrotransposal insertion in a non-Japanese Fukuyama congenital muscular dystrophy (FCMD) patient.

  Hui Xiong, Shuo Wang, Kazuhiro Kobayashi, Yuwu Jiang, Jingmin Wang, Xingzhi Chang, Yun Yuan, Jieyu Liu, Tatsushi Toda, Yukio Fukuyama, Xiru Wu
  [show abstract] [hide abstract]
  ABSTRACT: Fukuyama-type congenital muscular dystrophy (FCMD) is an autosomal recessive disorder, characterized by severe muscular dystrophy associated with brain malformation. FCMD is the second most common form of muscular dystrophy and one of the most common autosomal recessive diseases among the Japanese population; however, no typical FCMD cases have been reported in any other population. In this study, we report on the first identification of a Chinese FCMD patient; our findings are supported by clinical, histological, and magnetic resonance imaging (MRI) evidence, as well as fukutin gene mutational analyses. The patient presented with neonatal hypotonia, seizures, and delayed motor and speech development. Additional testing revealed cerebral and cerebellar gyrus abnormalities with white matter signal intensity changes, elevated serum creatine kinase (CK) levels, and dystrophic skeletal muscle with alpha-dystroglycan hypoglycosylation, and normal beta-dystroglycan and merosin expression. Genetic analysis of the fukutin gene showed one copy with a Japanese founder 3-kilobase (kb) retrotransposal insertion in the 3'-non-coding region and the other copy with a known c.139C>T mutation. This is the first FCMD case reported in the Chinese population and the first case in which the 3-kb insertion has been found outside of the Japanese population. This report emphasizes the importance of considering the fukutin founder mutation for diagnostic purposes outside of Japan.
  American Journal of Medical Genetics Part A 11/2009; 149A(11):2403-8. · 2.39 Impact Factor
• Article: Spinocerebellar ataxia type 31 is associated with "inserted" penta-nucleotide repeats containing (TGGAA)n.

  Nozomu Sato, Takeshi Amino, Kazuhiro Kobayashi, Shuichi Asakawa, Taro Ishiguro, Taiji Tsunemi, Makoto Takahashi, Tohru Matsuura, Kevin M Flanigan, Sawa Iwasaki, [......], Yuko Saito, Shigeo Murayama, Mari Yoshida, Yoshio Hashizume, Yuji Takahashi, Shoji Tsuji, Nobuyoshi Shimizu, Tatsushi Toda, Kinya Ishikawa, Hidehiro Mizusawa
  [show abstract] [hide abstract]
  ABSTRACT: Spinocerebellar ataxia type 31 (SCA31) is an adult-onset autosomal-dominant neurodegenerative disorder showing progressive cerebellar ataxia mainly affecting Purkinje cells. The SCA31 critical region was tracked down to a 900 kb interval in chromosome 16q22.1, where the disease shows a strong founder effect. By performing comprehensive Southern blot analysis and BAC- and fosmid-based sequencing, we isolated two genetic changes segregating with SCA31. One was a single-nucleotide change in an intron of the thymidine kinase 2 gene (TK2). However, this did not appear to affect splicing or expression patterns. The other was an insertion, from 2.5-3.8 kb long, consisting of complex penta-nucleotide repeats including a long (TGGAA)n stretch. In controls, shorter (1.5-2.0 kb) insertions lacking (TGGAA)n were found only rarely. The SCA31 repeat insertion's length inversely correlated with patient age of onset, and an expansion was documented in a single family showing anticipation. The repeat insertion was located in introns of TK2 and BEAN (brain expressed, associated with Nedd4) expressed in the brain and formed RNA foci in the nuclei of patients' Purkinje cells. An electrophoretic mobility-shift assay showed that essential splicing factors, serine/arginine-rich splicing factors SFRS1 and SFRS9, bind to (UGGAA)n in vitro. Because (TGGAA)n is a characteristic sequence of paracentromeric heterochromatin, we speculate that the insertion might have originated from heterochromatin. SCA31 is important because it exemplifies human diseases associated with "inserted" microsatellite repeats that can expand through transmission. Our finding suggests that the ectopic microsatellite repeat, when transcribed, might cause a disease involving the essential splicing factors.
  The American Journal of Human Genetics 10/2009; 85(5):544-57. · 10.60 Impact Factor
• Source

  Available from: Yuko Miyagoe-Suzuki

  Article: Residual laminin-binding activity and enhanced dystroglycan glycosylation by LARGE in novel model mice to dystroglycanopathy.

  Motoi Kanagawa, Akemi Nishimoto, Tomohiro Chiyonobu, Satoshi Takeda, Yuko Miyagoe-Suzuki, Fan Wang, Nobuhiro Fujikake, Mariko Taniguchi, Zhongpeng Lu, Masaji Tachikawa, Yoshitaka Nagai, Fumi Tashiro, Jun-Ichi Miyazaki, Youichi Tajima, Shin'ichi Takeda, Tamao Endo, Kazuhiro Kobayashi, Kevin P Campbell, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: Hypoglycosylation and reduced laminin-binding activity of alpha-dystroglycan are common characteristics of dystroglycanopathy, which is a group of congenital and limb-girdle muscular dystrophies. Fukuyama-type congenital muscular dystrophy (FCMD), caused by a mutation in the fukutin gene, is a severe form of dystroglycanopathy. A retrotransposal insertion in fukutin is seen in almost all cases of FCMD. To better understand the molecular pathogenesis of dystroglycanopathies and to explore therapeutic strategies, we generated knock-in mice carrying the retrotransposal insertion in the mouse fukutin ortholog. Knock-in mice exhibited hypoglycosylated alpha-dystroglycan; however, no signs of muscular dystrophy were observed. More sensitive methods detected minor levels of intact alpha-dystroglycan, and solid-phase assays determined laminin binding levels to be approximately 50% of normal. In contrast, intact alpha-dystroglycan is undetectable in the dystrophic Large(myd) mouse, and laminin-binding activity is markedly reduced. These data indicate that a small amount of intact alpha-dystroglycan is sufficient to maintain muscle cell integrity in knock-in mice, suggesting that the treatment of dystroglycanopathies might not require the full recovery of glycosylation. To examine whether glycosylation defects can be restored in vivo, we performed mouse gene transfer experiments. Transfer of fukutin into knock-in mice restored glycosylation of alpha-dystroglycan. In addition, transfer of LARGE produced laminin-binding forms of alpha-dystroglycan in both knock-in mice and the POMGnT1 mutant mouse, which is another model of dystroglycanopathy. Overall, these data suggest that even partial restoration of alpha-dystroglycan glycosylation and laminin-binding activity by replacing or augmenting glycosylation-related genes might effectively deter dystroglycanopathy progression and thus provide therapeutic benefits.
  Human Molecular Genetics 12/2008; 18(4):621-31. · 7.64 Impact Factor
• Article: Reduced expression of sarcospan in muscles of Fukuyama congenital muscular dystrophy.

  Yoshihiro Wakayama, Masahiko Inoue, Hiroko Kojima, Sumimasa Yamashita, Seiji Shibuya, Takahiro Jimi, Hajime Hara, Yoko Matsuzaki, Hiroaki Oniki, Motoi Kanagawa, Kazuhiro Kobayashi, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: Expression profiles of sarcospan in muscles with muscular dystrophies are scarcely reported. To examine this, we studied five Fukuyama congenital muscular dystrophy (FCMD) muscles, five Duchenne muscular dystrophy (DMD) muscles, five disease control and five normal control muscles. Immunoblot showed reactions of sarcospan markedly decreased in FCMD and DMD muscle extracts. Immunohistochemistry of FCMD muscles showed that most large diameter myofibers expressed sarcospan discontinuously at their surface membranes. Immature small diameter FCMD myofibers usually did not express sarcospan. Immunoreactivity of sarcospan in DMD muscles was similarly reduced. With regard to dystroglycans and sarcoglycans, immunohistochemistry of FCMD muscles showed selective deficiency of glycosylated alpha-dystroglycan, together with reduced expression of beta-dystroglycan and alpha-, beta-, gamma-, delta-sarcoglycans. Although the expression of glycosylated alpha-dystroglycan was lost, scattered FCMD myofibers showed positive immunoreaction with an antibody against the core protein of alpha-dystroglycan. The group mean ratios of sarcospan mRNA copy number versus GAPDH mRNA copy number by real-time RT-PCR showed that the ratios between FCMD and normal control groups were not significantly different (P>0.1 by the two-tailed t test). This study implied either O-linked glycosylation defects of alpha-dystroglycan in the Golgi apparatus of FCMD muscles may lead to decreased expression of sarcoglycan and sarcospan molecules, or selective deficiency of glycosylated alpha-dystroglycan due to impaired glycosylation in FCMD muscles may affect the molecular integrity of the basal lamina of myofibers. This, in turn, leads to decreased expression of sarcoglycans, and finally of sarcospan at the FCMD myofiber surfaces.
  Histology and histopathology 12/2008; 23(12):1425-38. · 2.48 Impact Factor
• Source

  Available from: bris.ac.uk

  Article: Pikachurin, a dystroglycan ligand, is essential for photoreceptor ribbon synapse formation.

  Shigeru Sato, Yoshihiro Omori, Kimiko Katoh, Mineo Kondo, Motoi Kanagawa, Kentaro Miyata, Kazuo Funabiki, Toshiyuki Koyasu, Naoko Kajimura, Tomomitsu Miyoshi, Hajime Sawai, Kazuhiro Kobayashi, Akiko Tani, Tatsushi Toda, Jiro Usukura, Yasuo Tano, Takashi Fujikado, Takahisa Furukawa
  [show abstract] [hide abstract]
  ABSTRACT: Exquisitely precise synapse formation is crucial for the mammalian CNS to function correctly. Retinal photoreceptors transfer information to bipolar and horizontal cells at a specialized synapse, the ribbon synapse. We identified pikachurin, an extracellular matrix-like retinal protein, and observed that it localized to the synaptic cleft in the photoreceptor ribbon synapse. Pikachurin null-mutant mice showed improper apposition of the bipolar cell dendritic tips to the photoreceptor ribbon synapses, resulting in alterations in synaptic signal transmission and visual function. Pikachurin colocalized with both dystrophin and dystroglycan at the ribbon synapses. Furthermore, we observed direct biochemical interactions between pikachurin and dystroglycan. Together, our results identify pikachurin as a dystroglycan-interacting protein and demonstrate that it has an essential role in the precise interactions between the photoreceptor ribbon synapse and the bipolar dendrites. This may also advance our understanding of the molecular mechanisms underlying the retinal electrophysiological abnormalities observed in muscular dystrophy patients.
  Nature Neuroscience 08/2008; 11(8):923-31. · 15.53 Impact Factor
• Article: Partial tandem duplication of GRIA3 in a male with mental retardation.

  Tomohiro Chiyonobu, Shin Hayashi, Kazuhiro Kobayashi, Masafumi Morimoto, Yuri Miyanomae, Akira Nishimura, Akemi Nishimoto, Chiyomi Ito, Issei Imoto, Tohru Sugimoto, Zhengping Jia, Johji Inazawa, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: The genetic factors underlying mental retardation (MR) are very heterogeneous. Recent studies have identified a number of genes involved in MR, several of which lie on the X-chromosome, but the current understanding of the monogenic causes of MR is far from complete. Investigation of chromosomal rearrangements in patients with MR has proven particularly informative in the search for novel genes. Using array-based comparative genomic hybridization analysis, we identified a small copy number gain at Xq25, which was undetectable by conventional G-band analysis, in a boy with unexplained MR. Further characterization revealed a partial tandem duplication of GRIA3, an alteration also present on one allele in his mother. RT-PCR analysis of lymphoblastoid cell RNA revealed remarkably reduced GRIA3 transcript levels in the patient. The mother, whose cognitive level is normal, also demonstrated remarkably reduced GRIA3 transcript levels in lymphoblastoid cells, and X-chromosome inactivation (XCI) was completely skewed in her peripheral lymphocytes. It is possible that XCI in the brain is not completely skewed and that GRIA3 expression from the normal allele may account for the mother's normal cognitive function. Taken together with previous findings of GRIA3 disruptions in the patients with MR, our study strengthens the idea that GRIA3 is a candidate gene for X-linked MR and that severely reduced GRIA3 expression results in MR.
  American Journal of Medical Genetics Part A 08/2007; 143A(13):1448-55. · 2.39 Impact Factor
• Article: Molecular interaction between fukutin and POMGnT1 in the glycosylation pathway of alpha-dystroglycan.

  Hui Xiong, Kazuhiro Kobayashi, Masaji Tachikawa, Hiroshi Manya, Satoshi Takeda, Tomohiro Chiyonobu, Nobuhiro Fujikake, Fan Wang, Akemi Nishimoto, Glenn E Morris, Yoshitaka Nagai, Motoi Kanagawa, Tamao Endo, Tatsushi Toda
  [show abstract] [hide abstract]
  ABSTRACT: The recent identification of mutations in genes encoding demonstrated or putative glycosyltransferases has revealed a novel mechanism for congenital muscular dystrophy. Hypoglycosylated alpha-dystroglycan (alpha-DG) is commonly seen in Fukuyama-type congenital muscular dystrophy (FCMD), muscle-eye-brain disease (MEB), Walker-Warburg syndrome (WWS), and Large(myd) mice. POMGnT1 and POMTs, the gene products responsible for MEB and WWS, respectively, synthesize unique O-mannose sugar chains on alpha-DG. The function of fukutin, the gene product responsible for FCMD, remains undetermined. Here we show that fukutin co-localizes with POMGnT1 in the Golgi apparatus. Direct interaction between fukutin and POMGnT1 was confirmed by co-immunoprecipitation and two-hybrid analyses. The transmembrane region of fukutin mediates its localization to the Golgi and participates in the interaction with POMGnT1. Y371C, a missense mutation found in FCMD, retains fukutin in the ER and also redirects POMGnT1 to the ER. Finally, we demonstrate reduced POMGnT1 enzymatic activity in transgenic knock-in mice carrying the retrotransposal insertion in the fukutin gene, the prevalent mutation in FCMD. From these findings, we propose that fukutin forms a complex with POMGnT1 and may modulate its enzymatic activity.
  Biochemical and Biophysical Research Communications 01/2007; 350(4):935-41. · 2.48 Impact Factor
• Article: Redefining the disease locus of 16q22.1-linked autosomal dominant cerebellar ataxia.

  Takeshi Amino, Kinya Ishikawa, Shuta Toru, Taro Ishiguro, Nozomu Sato, Taiji Tsunemi, Miho Murata, Kazuhiro Kobayashi, Johji Inazawa, Tatsushi Toda, Hidehiro Mizusawa
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  ABSTRACT: The 16q22.1-linked autosomal dominant cerebellar ataxia (16q-ADCA; Online Mendelian Inheritance in Man [OMIN] #117210) is one of the most common ADCAs in Japan. Previously, we had reported that the patients share a common haplotype by founder effect and that a C-to-T substitution (-16C>T) in the puratrophin-1 gene was strongly associated with the disease. However, recently, an exceptional patient without the substitution was reported, indicating that a true pathogenic mutation might be present elsewhere. In this study, we clarified the disease locus more definitely by the haplotype analysis of families showing pure cerebellar ataxia. In addition to microsatellite markers, the single nucleotide polymorphisms (SNPs) that we identified on the disease chromosome were examined to confirm the borders of the disease locus. The analysis of 64 families with the -16C>T substitution in the puratrophin-1 gene revealed one family showing an ancestral recombination event between SNP04 and SNP05 on the disease chromosome. The analysis of 22 families without identifiable genetic mutations revealed another family carrying the common haplotype centromeric to the puratrophin-1 gene, but lacking the -16C>T substitution in this gene. We concluded that the disease locus of 16q-ADCA was definitely confined to a 900-kb genomic region between the SNP04 and the -16C>T substitution in the puratrophin-1 gene in 16q22.1.
  Journal of Human Genetics 01/2007; 52(8):643-9. · 2.57 Impact Factor
• Article: Founder SVA retrotransposal insertion in Fukuyama-type congenital muscular dystrophy and its origin in Japanese and Northeast Asian populations.

  Masashi Watanabe, Kazuhiro Kobayashi, Feng Jin, Kyung Sook Park, Takatsugu Yamada, Katsushi Tokunaga, Tatsushi Toda
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  ABSTRACT: Fukuyama-type congenital muscular dystrophy (FCMD), one of the most common autosomal recessive disorders in Japan, is characterized by congenital muscular dystrophy associated with brain malformation due to a defect in neuronal migration. Previously, we identified the gene responsible for FCMD, which encodes the fukutin protein. Most FCMD-bearing chromosomes (87%) are derived from a single ancestral founder, who lived 2,000-2,500 years ago and whose mutation consisted of a 3-kb retrotransposal insertion in the 3' non-coding region of the fukutin gene. Here we show, through detailed sequence analysis, that the founder insertion is derived from the SINE-VNTR-Alu (SVA) retroposon. To enable rapid detection of this insertion, we have developed a PCR-based diagnostic method that uses three primers simultaneously. We used this method to investigate the distribution and origin of the founder insertion, screening a total of 4,718 control DNA samples from Japanese and other Northeast Asian populations. Fifteen founder chromosomes were detected among 2,814 Japanese individuals. Heterozygous carriers were found in various regions throughout Japan, with an averaged ratio of 1 in 188. In Korean populations, we detected one carrier in 935 individuals. However, we were unable to detect any heterozygous alleles in 203 Mongolians and 766 Mainland Chinese populations. These data largely rule out the possibility that a single ancestor bearing an insertion-chromosome immigrated to Japan from Korea or Mainland China and appear to confirm that FCMD carriers are rare outside of Japan.
  American Journal of Medical Genetics Part A 12/2005; 138(4):344-8. · 2.39 Impact Factor
• Article: beta4GalT-II is a key regulator of glycosylation of the proteins involved in neuronal development.

  Norihiko Sasaki, Hiroshi Manya, Reiko Okubo, Kazuhiro Kobayashi, Hideki Ishida, Tatsushi Toda, Tamao Endo, Shoko Nishihara
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  ABSTRACT: Seven members of the human beta1,4-galactosyltransferase (beta4GalTs) have been identified and characterized by many groups. beta4GalTs play important roles in the extension of N- and O-linked glycans involved in several biological events. However, it has not been clear which beta4GalTs can act on glycoproteins, such as alpha-dystroglycan and Notch receptors, involved in neuronal development. To clarify which beta4GalTs can function, we determined the enzyme activities toward such motifs and the transcript levels in human normal tissues. Among human beta4GalTs, both beta4GalT-I and beta4GalT-II could act efficiently on all substrates, but the relative activity of beta4GalT-II was higher than that of beta4GalT-I. Transcript of beta4GalT-I was widely expressed except for brain, and on the other hand, that of beta4GalT-II was expressed at high levels in the brain. Thus, these results suggest that among human beta4GalTs, beta4GalT-II is a major regulator of the synthesis of glycans involved in neuronal development.
  Biochemical and Biophysical Research Communications 08/2005; 333(1):131-7. · 2.48 Impact Factor