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G.P.2.11 Developmental defects in a zebrafish model for muscular dystrophies associated with the loss of FKRP
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Abstracts / Neuromuscular Disorders 18 (2008) 724–833
lines such as HeLa or MCF7 did not. Using an antibody against core protein, a-DG with molecular mass of 75 kDa was detected in these carcinoma cells, suggesting that functional a-dystroglycan is lost in carcinoma cells. Next, we examined if the function of a-DG is restored in these cells by gene transfer of glycosyltransferases, mutation of which leads to a-dystroglycanopathy. Transfection of the cells with LARGE greatly restored the laminin binding activity of a-DG, whereas other glycosyltransferases including POMGnT1, POMT1, POMT2, fukutin or FKRP did not. These results demonstrate that the restoration of functional a-DG by LARGE is a possible molecular target when developing therapeutic strategies for a-dystroglycanopathy. doi:10.1016/j.nmd.2008.06.049
G.P.2.10 Characterisation of the brain and eye phenotype of a FKRP knock-down mouse model of Muscle–Eye–Brain disease M.R. Ackroyd 1; L. Skordis 1; M. Kaluarachchi 1; S. Prior 1; F. Muntoni 2; S.C. Brown 1 1 Imperial College, Department of Neuroscience, London, United Kingdom; 2 Institute of Child Health, University College London, London, United Kingdom Mutations in the gene encoding for fukutin related protein (FKRP) are responsible for a spectrum of disorders that are classified within the group now known as the dystroglycanopathies. These range from limb-girdle muscular dystrophies through to Muscle–Eye–Brain disease (MEB) and Walker–Warburg syndrome (WWS). We have created a novel mouse model with reduced FKRP expression that resembles MEB and has severe structural brain defects indicative of disordered neuronal migration. Nuclei counts across the cerebral cortex illustrate only a slight reduction in overall number in the mutant compared to wild type but a significant disruption in the later formed cortical layers where there were significantly fewer nuclei in affected mice. Immunolabelling with the neuronal marker NeuN which identifies a population of post mitotic neurons confirmed that many of these mis-placed nuclei were of neuronal origin. These data are consistent with a defective pial basement membrane resulting from the hypoglycosylation of alpha-dystroglycan which subsequently disrupts the radial glial scaffold. We have now shown that there is a similar disruption in the cerebellar basement membrane and will present BrdU birth dating together with immunocytochemical analyses to compare modes of neuronal migration in the cortex and cerebellum. We have also identified a disruption of the inner limiting basement membrane of the eye with an associated alteration in positioning of the ganglion cell layer. Detailed immunocytochemical studies suggest that this is due to a novel interaction between alpha-dystroglycan and components of the extracellular matrix. These studies identify some of the key pathological mechanisms of disease associated with a reduction in the expression of FKRP. doi:10.1016/j.nmd.2008.06.050
G.P.2.11
burg syndrome (WWS), which are associated with brain and eye abnormalities. The defective glycosylation of a-dystroglycan in these disorders leads to a failure of a-dystroglycan to bind to extracellular matrix components and previous attempts to model these disorders have shown that the generation of fukutin and Pomt1 deficient knockout mice results in early embryonic lethality due to basement membrane defects. We have used the zebrafish as an animal model to investigate the pathological consequences of down-regulating the expression of the putative glycosyltransferase gene fukutin-related protein (FKRP) on embryonic development. We have found that down-regulating FKRP in the zebrafish results in embryos which develop a range of abnormalities reminiscent of the developmental defects observed in human muscular dystrophies associated with mutations in the FKRP gene. FKRP morphant embryos showed a spectrum of phenotypic severity involving alterations in somitic structure and muscle fibre organisation as well as defects in developing neuronal structures and eye morphology. The pathological phenotype was found to correlate with a reduction in a-dystroglycan glycosylation and reduced laminin binding. Further characterisation of the developmental processes affected in FKRP morphant embryos may lead to a better understanding of the pathological spectrum observed in muscular dystrophies associated with mutations in the human FKRP gene. doi:10.1016/j.nmd.2008.06.051
G.P.2.12 Generation of a model mouse for Fukuyama congenital muscular dystrophy carrying a retrotransposal insertion in the 30 UTR in the fukutin gene: Therapeutic benefit of enhanced dystroglycan glycosylation to dystroglycanopathy T. Toda 1; M. Kanagawa 1; A. Nishimoto 1; T. Chiyonobu 1; S. Takeda 2 1 Osaka University Graduate School of Medicine, Division of Clinical Genetics, Suita, Osaka, Japan; 2 Otsuka GEN Research Institute, Tokushima, Japan Fukuyama congenital muscular dystrophy (FCMD) is characterized by severe congenital muscular dystrophy with brain anomaly. FCMD is caused by mutations in fukutin and the major mutation is a retrotransposal insertion in the 30 untranslated region. In FCMD, dystroglycan (DG), a cell surface receptor for matrix proteins, is hypoglycosylated and loses the laminin-binding activity. To understand the pathogenesis and develop a therapeutic strategy, we generated a model mouse carrying the retrotransposal insertion. The targeting vector was generated using mutant lox system. Exon 10 of mouse fukutin was substituted by exon 10 of human patient’s fukutin with the retrotransposal insertion. Mice homozygous for the mutation show no typical sign of muscular dystrophy. Western blotting analysis shows that the majority of DG species are hypoglycosylated but functionally glycosylated forms are also present. Solid-phase binding assays indicated that more than 50% of laminin-binding activity remained in the mutant skeletal muscle. Finally, we show that abnormally glycosylated DG species were restored after adenoviral gene transfer of the fukutin gene. Taken together, these data demonstrate that the presence of only a small population of functionally glycosylated DG is sufficient to prevent disease progression, suggesting a possibility of glycotherapy to a group of congenital muscular dystrophy.
Developmental defects in a zebrafish model for muscular dystrophies associated with the loss of FKRP P. Thornhill; D. Bassett; H. Lochmu¨ller; K. Bushby; V. Straub Newcastle University, Institute of Human Genetics, Newcastle upon Tyne, United Kingdom
doi:10.1016/j.nmd.2008.06.052
A number of muscular dystrophies (MD) are associated with the defective glycosylation of a-dystroglycan and many of which are now known to result from mutations in a number of genes encoding putative or known glycosyltransferases. These diseases include severe forms of congenital muscular dystrophy (CMD) such as Fukuyama type congenital muscular dystrophy (FCMD), Muscle–Eye–Brain disease (MEB) and Walker–War-
T.P.1.01
CLINICAL ASSESSMENT TOOLS; POSTER PRESENTATIONS
Use of the 6 min walk test as an endpoint in clinical trials for neuromuscular diseases J.M. Florence 1; A. van der Ploeg 2; P.R. Clemens 3; D.M. Escolar 4; P. Laforet 5; B. Rosenbloom 6; M. Wasserstein 7; A. Skrinar 8; A. Pestronk 1; J.E. Mayhew 4
Abstracts / Neuromuscular Disorders 18 (2008) 724–833
lines such as HeLa or MCF7 did not. Using an antibody against core protein, a-DG with molecular mass of 75 kDa was detected in these carcinoma cells, suggesting that functional a-dystroglycan is lost in carcinoma cells. Next, we examined if the function of a-DG is restored in these cells by gene transfer of glycosyltransferases, mutation of which leads to a-dystroglycanopathy. Transfection of the cells with LARGE greatly restored the laminin binding activity of a-DG, whereas other glycosyltransferases including POMGnT1, POMT1, POMT2, fukutin or FKRP did not. These results demonstrate that the restoration of functional a-DG by LARGE is a possible molecular target when developing therapeutic strategies for a-dystroglycanopathy. doi:10.1016/j.nmd.2008.06.049
G.P.2.10 Characterisation of the brain and eye phenotype of a FKRP knock-down mouse model of Muscle–Eye–Brain disease M.R. Ackroyd 1; L. Skordis 1; M. Kaluarachchi 1; S. Prior 1; F. Muntoni 2; S.C. Brown 1 1 Imperial College, Department of Neuroscience, London, United Kingdom; 2 Institute of Child Health, University College London, London, United Kingdom Mutations in the gene encoding for fukutin related protein (FKRP) are responsible for a spectrum of disorders that are classified within the group now known as the dystroglycanopathies. These range from limb-girdle muscular dystrophies through to Muscle–Eye–Brain disease (MEB) and Walker–Warburg syndrome (WWS). We have created a novel mouse model with reduced FKRP expression that resembles MEB and has severe structural brain defects indicative of disordered neuronal migration. Nuclei counts across the cerebral cortex illustrate only a slight reduction in overall number in the mutant compared to wild type but a significant disruption in the later formed cortical layers where there were significantly fewer nuclei in affected mice. Immunolabelling with the neuronal marker NeuN which identifies a population of post mitotic neurons confirmed that many of these mis-placed nuclei were of neuronal origin. These data are consistent with a defective pial basement membrane resulting from the hypoglycosylation of alpha-dystroglycan which subsequently disrupts the radial glial scaffold. We have now shown that there is a similar disruption in the cerebellar basement membrane and will present BrdU birth dating together with immunocytochemical analyses to compare modes of neuronal migration in the cortex and cerebellum. We have also identified a disruption of the inner limiting basement membrane of the eye with an associated alteration in positioning of the ganglion cell layer. Detailed immunocytochemical studies suggest that this is due to a novel interaction between alpha-dystroglycan and components of the extracellular matrix. These studies identify some of the key pathological mechanisms of disease associated with a reduction in the expression of FKRP. doi:10.1016/j.nmd.2008.06.050
G.P.2.11
burg syndrome (WWS), which are associated with brain and eye abnormalities. The defective glycosylation of a-dystroglycan in these disorders leads to a failure of a-dystroglycan to bind to extracellular matrix components and previous attempts to model these disorders have shown that the generation of fukutin and Pomt1 deficient knockout mice results in early embryonic lethality due to basement membrane defects. We have used the zebrafish as an animal model to investigate the pathological consequences of down-regulating the expression of the putative glycosyltransferase gene fukutin-related protein (FKRP) on embryonic development. We have found that down-regulating FKRP in the zebrafish results in embryos which develop a range of abnormalities reminiscent of the developmental defects observed in human muscular dystrophies associated with mutations in the FKRP gene. FKRP morphant embryos showed a spectrum of phenotypic severity involving alterations in somitic structure and muscle fibre organisation as well as defects in developing neuronal structures and eye morphology. The pathological phenotype was found to correlate with a reduction in a-dystroglycan glycosylation and reduced laminin binding. Further characterisation of the developmental processes affected in FKRP morphant embryos may lead to a better understanding of the pathological spectrum observed in muscular dystrophies associated with mutations in the human FKRP gene. doi:10.1016/j.nmd.2008.06.051
G.P.2.12 Generation of a model mouse for Fukuyama congenital muscular dystrophy carrying a retrotransposal insertion in the 30 UTR in the fukutin gene: Therapeutic benefit of enhanced dystroglycan glycosylation to dystroglycanopathy T. Toda 1; M. Kanagawa 1; A. Nishimoto 1; T. Chiyonobu 1; S. Takeda 2 1 Osaka University Graduate School of Medicine, Division of Clinical Genetics, Suita, Osaka, Japan; 2 Otsuka GEN Research Institute, Tokushima, Japan Fukuyama congenital muscular dystrophy (FCMD) is characterized by severe congenital muscular dystrophy with brain anomaly. FCMD is caused by mutations in fukutin and the major mutation is a retrotransposal insertion in the 30 untranslated region. In FCMD, dystroglycan (DG), a cell surface receptor for matrix proteins, is hypoglycosylated and loses the laminin-binding activity. To understand the pathogenesis and develop a therapeutic strategy, we generated a model mouse carrying the retrotransposal insertion. The targeting vector was generated using mutant lox system. Exon 10 of mouse fukutin was substituted by exon 10 of human patient’s fukutin with the retrotransposal insertion. Mice homozygous for the mutation show no typical sign of muscular dystrophy. Western blotting analysis shows that the majority of DG species are hypoglycosylated but functionally glycosylated forms are also present. Solid-phase binding assays indicated that more than 50% of laminin-binding activity remained in the mutant skeletal muscle. Finally, we show that abnormally glycosylated DG species were restored after adenoviral gene transfer of the fukutin gene. Taken together, these data demonstrate that the presence of only a small population of functionally glycosylated DG is sufficient to prevent disease progression, suggesting a possibility of glycotherapy to a group of congenital muscular dystrophy.
Developmental defects in a zebrafish model for muscular dystrophies associated with the loss of FKRP P. Thornhill; D. Bassett; H. Lochmu¨ller; K. Bushby; V. Straub Newcastle University, Institute of Human Genetics, Newcastle upon Tyne, United Kingdom
doi:10.1016/j.nmd.2008.06.052
A number of muscular dystrophies (MD) are associated with the defective glycosylation of a-dystroglycan and many of which are now known to result from mutations in a number of genes encoding putative or known glycosyltransferases. These diseases include severe forms of congenital muscular dystrophy (CMD) such as Fukuyama type congenital muscular dystrophy (FCMD), Muscle–Eye–Brain disease (MEB) and Walker–War-
T.P.1.01
CLINICAL ASSESSMENT TOOLS; POSTER PRESENTATIONS
Use of the 6 min walk test as an endpoint in clinical trials for neuromuscular diseases J.M. Florence 1; A. van der Ploeg 2; P.R. Clemens 3; D.M. Escolar 4; P. Laforet 5; B. Rosenbloom 6; M. Wasserstein 7; A. Skrinar 8; A. Pestronk 1; J.E. Mayhew 4