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Neuromuscular junction biology
Poster Presentations Design/Methods: Thirty open biopsied samples of biceps muscle were analyzed from 7 DM1 patients, 9 non-DM1 myopathic disease controls (6 limb-girdle and 3 facioscapulohumeral muscular dystrophy patients) and 5 normal individuals exhibiting no pathological findings. Results: One mRNA out of at least 18,000 RT-PCR products expressed differentially between patients with DM1 and normal individuals, which encoded the CUG binding protein 1 (CUGBP1). Amount of CUGBP1 mRNA, as well as CUGBP1 protein, was reduced in biopsies from DM1 patients, relative to samples from myopathic disease controls and normal individuals. Conclusions: Present screen has revealed that the expression of CUGBP1 was altered in patients with DM1.
P-679 Myotonic Dystrophy Type 1: Clinical and Molecular Analysis in 12 Families from Uruguay D. Yorio* 1, D. Garc a-Arocena 1, D. Pel ez 1, C. Braidal, M. Rodr guez Teja I , M.M. Rodr guez I , J.P. McAbney 2, D. Monckton 2, M. Medici 1.
1Montevideo, Uruguay; 2Glasgow, UK Objective: The genetic basis of Myotonic Dystrophy tipe 1 (DM1) is a dynamic CTG expansion in the DMPK gene on chromosome 19. As clinical identification of mild cases can be difficult, it was our aim to develop molecular analysis for DM1 in Uruguay in order to confirm clinical diagnosis in affected patients and rule out the disease in at risk relatives. Methods: Twelve unrelated patients and 20 at risk relatives were clinically examined and the CTG repeat length determined by Southern blot analysis and/or polymerase chain reaction (PCR). Results: Patients showed a wide variation in the range and severity of muscular and systemic signs, with a high prevalence of thyroid abnormalities. The clinical diagnosis of DM1 was confirmed by molecular analysis in 21/22 cases. Southern blot analysis showed large expansions ranging from 130 to 3610 repeats in 17 patients while PCR detected 4 carriers of small expansions in the low end of disease range. The patient with DM1 phenotype and normal molecular analysis will be discussed. Conclusions: We found no significant correlation between the number of CTG repeats and the clinical manifestations in these families, probably due to the small number of patients examined and the confounding effects of age dependent somatic mosaicism on measured allele length.
Neuromuscular Junction Biology P-680 Perlecan is Essentail for Clustering of Acetylcholinesterase at the Neuromuscular Junction E. Arikawa-Hirasawa* 1,3, S.G. Rossi 2, K.Sugie 3, R.L. Rotundo 2, Y. Yamada t . 1Bethesda, USA; 2Miami, USA; 3Tokyo, Japan
Objective: Perlecan is a multifunctional heparan sulfate proteoglycan expressed in all basement membranes and other matrices such as cartilage. Perlecan is enriched at the neuromuscularjunction (NMJ) and co-localizes with acetylcholinesterase (ACHE). Functional null mutations of the perlecan gene in humans and mice cause lethal skeletal dysplasia. Perlecan mutations were also identified in the relatively mild chondrodystrophic myotonia, Schwartz-Jampel syndrome (SJS). Methods: In order to define the role of perlecan in NMJ function, we examined molecules clustering at the NMJ of perlecan-null mice. Results: We demonstrated that most of the molecules localized at the normal NMJ, such as acetylcholine receptor (AChR), alpha- and betadystroglycans, utrophin, rapsin, and agrin, were concentrated at the NMJ of perlecan-null mice. However, AChE was completely absent at the perlecan-null NMJ, whereas it was co-localized with perlecan and other clustering molecules at the normal NMJ. Conclusions: Thus, perlecan is the unique acceptor molecule for AChE at the NMJ. Our results provide the molecular mechanism of how AChE becomes localized at the synapse.
Thursday, July 11, 2002
S109
P-681 Expression of a PDZ-Deleted D L G in Skeletal Muscle of Transgenic Mice Results in Alterations in Receptors Clustering and Neuromuscular Junction Abnormalities T.A. Hainsey, J.A. Rafael. Columbus, OH, USA
Objective: Determine the role of the Dlg PDZ domains on receptor and channel clustering at the neuromuscular junction. Methods: Western blot and immunohistochemical analyses of PDZdeleted Dlg transgenic mice show the expression and localization of Dlg and possible interactors. Immunoprecipitations of Dlg from membrane preparations of C57 quadriceps muscles reveal interacting proteins, receptors, and channels. Results: Dlg is thought to play an important role in clustering channels and receptors at brain synapses, hut its function in muscle is unknown. A PDZ domain deleted version of Dlg is expressed in skeletal muscle and replaces the endogenous form of the protein. Deletion of the PDZ domain does not cause mislocalization of the protein. Preliminary data suggests that Dlg interacts with ErbB4 Receptors, NMDA Receptor 2A, and another PDZ domain-containing protein, CASK; and that there are differences in the levels and localization of ErbB4 and NMDA R2A receptors in transgenic quadriceps muscles. Neuromuscular junctions in the quadriceps muscles of PDZ-deleted Dlg transgenic animals are significantly smaller and less branched than in wild type muscle. Conclusion: These data suggest that the PDZ domains of Dlg may play an important role in the clustering of proteins and receptors, as well as in the structure of the mammalian neuromuscular junction. P-682 CASK is a Component of a PDZ Domain-Containing Protein Scaffold at the Neuromuscular Junction J.L. Siders, J.A. Rafael. Columbus, OH, US
Objective: To determine the function of the PDZ domain-containing protein CASK in skeletal muscle. Methods: Immunohistochemical analysis, immunoprecipitations, and immunoblots of mouse quadriceps muscle were used to determine the localization, developmental expression and protein interactions of CASK in skeletal muscle. Results: CASK is thought to play an important role in receptor clustering at synapses in the brain, but its role in muscle is unknown. CASK is enriched in membrane preparations of C57 BL/10 quadriceps muscles and is specifically localized at the post-synaptic membrane of the neuromuscular junction as well as a subset of nuclei. In cultured C2C12 cells, CASK localization moves from the nucleus to the cytoplasm and its expression is upregulated as myoblasts differentiate into mature myofibers. Treatment of C2C 12 cells with agrin does not resuk in the co-clustering of CASK with acetylcholine receptor complexes. Grip, another PDZ domaincontaining protein, is also present at the post-synaptic membrane of the neuromuscular junction and interacts with CASK in vivo in skeletal muscle. Conclusions: CASK is a member of PDZ domain-containing protein scaffold at the post-synaptic membrane of the neuromuscular junction. CASK does not cluster in response to agrin-induced acetylcholine receptor clustering suggesting a novel mechanism of localization of CASK to the neuromuscular junction.
P-679 Myotonic Dystrophy Type 1: Clinical and Molecular Analysis in 12 Families from Uruguay D. Yorio* 1, D. Garc a-Arocena 1, D. Pel ez 1, C. Braidal, M. Rodr guez Teja I , M.M. Rodr guez I , J.P. McAbney 2, D. Monckton 2, M. Medici 1.
1Montevideo, Uruguay; 2Glasgow, UK Objective: The genetic basis of Myotonic Dystrophy tipe 1 (DM1) is a dynamic CTG expansion in the DMPK gene on chromosome 19. As clinical identification of mild cases can be difficult, it was our aim to develop molecular analysis for DM1 in Uruguay in order to confirm clinical diagnosis in affected patients and rule out the disease in at risk relatives. Methods: Twelve unrelated patients and 20 at risk relatives were clinically examined and the CTG repeat length determined by Southern blot analysis and/or polymerase chain reaction (PCR). Results: Patients showed a wide variation in the range and severity of muscular and systemic signs, with a high prevalence of thyroid abnormalities. The clinical diagnosis of DM1 was confirmed by molecular analysis in 21/22 cases. Southern blot analysis showed large expansions ranging from 130 to 3610 repeats in 17 patients while PCR detected 4 carriers of small expansions in the low end of disease range. The patient with DM1 phenotype and normal molecular analysis will be discussed. Conclusions: We found no significant correlation between the number of CTG repeats and the clinical manifestations in these families, probably due to the small number of patients examined and the confounding effects of age dependent somatic mosaicism on measured allele length.
Neuromuscular Junction Biology P-680 Perlecan is Essentail for Clustering of Acetylcholinesterase at the Neuromuscular Junction E. Arikawa-Hirasawa* 1,3, S.G. Rossi 2, K.Sugie 3, R.L. Rotundo 2, Y. Yamada t . 1Bethesda, USA; 2Miami, USA; 3Tokyo, Japan
Objective: Perlecan is a multifunctional heparan sulfate proteoglycan expressed in all basement membranes and other matrices such as cartilage. Perlecan is enriched at the neuromuscularjunction (NMJ) and co-localizes with acetylcholinesterase (ACHE). Functional null mutations of the perlecan gene in humans and mice cause lethal skeletal dysplasia. Perlecan mutations were also identified in the relatively mild chondrodystrophic myotonia, Schwartz-Jampel syndrome (SJS). Methods: In order to define the role of perlecan in NMJ function, we examined molecules clustering at the NMJ of perlecan-null mice. Results: We demonstrated that most of the molecules localized at the normal NMJ, such as acetylcholine receptor (AChR), alpha- and betadystroglycans, utrophin, rapsin, and agrin, were concentrated at the NMJ of perlecan-null mice. However, AChE was completely absent at the perlecan-null NMJ, whereas it was co-localized with perlecan and other clustering molecules at the normal NMJ. Conclusions: Thus, perlecan is the unique acceptor molecule for AChE at the NMJ. Our results provide the molecular mechanism of how AChE becomes localized at the synapse.
Thursday, July 11, 2002
S109
P-681 Expression of a PDZ-Deleted D L G in Skeletal Muscle of Transgenic Mice Results in Alterations in Receptors Clustering and Neuromuscular Junction Abnormalities T.A. Hainsey, J.A. Rafael. Columbus, OH, USA
Objective: Determine the role of the Dlg PDZ domains on receptor and channel clustering at the neuromuscular junction. Methods: Western blot and immunohistochemical analyses of PDZdeleted Dlg transgenic mice show the expression and localization of Dlg and possible interactors. Immunoprecipitations of Dlg from membrane preparations of C57 quadriceps muscles reveal interacting proteins, receptors, and channels. Results: Dlg is thought to play an important role in clustering channels and receptors at brain synapses, hut its function in muscle is unknown. A PDZ domain deleted version of Dlg is expressed in skeletal muscle and replaces the endogenous form of the protein. Deletion of the PDZ domain does not cause mislocalization of the protein. Preliminary data suggests that Dlg interacts with ErbB4 Receptors, NMDA Receptor 2A, and another PDZ domain-containing protein, CASK; and that there are differences in the levels and localization of ErbB4 and NMDA R2A receptors in transgenic quadriceps muscles. Neuromuscular junctions in the quadriceps muscles of PDZ-deleted Dlg transgenic animals are significantly smaller and less branched than in wild type muscle. Conclusion: These data suggest that the PDZ domains of Dlg may play an important role in the clustering of proteins and receptors, as well as in the structure of the mammalian neuromuscular junction. P-682 CASK is a Component of a PDZ Domain-Containing Protein Scaffold at the Neuromuscular Junction J.L. Siders, J.A. Rafael. Columbus, OH, US
Objective: To determine the function of the PDZ domain-containing protein CASK in skeletal muscle. Methods: Immunohistochemical analysis, immunoprecipitations, and immunoblots of mouse quadriceps muscle were used to determine the localization, developmental expression and protein interactions of CASK in skeletal muscle. Results: CASK is thought to play an important role in receptor clustering at synapses in the brain, but its role in muscle is unknown. CASK is enriched in membrane preparations of C57 BL/10 quadriceps muscles and is specifically localized at the post-synaptic membrane of the neuromuscular junction as well as a subset of nuclei. In cultured C2C12 cells, CASK localization moves from the nucleus to the cytoplasm and its expression is upregulated as myoblasts differentiate into mature myofibers. Treatment of C2C 12 cells with agrin does not resuk in the co-clustering of CASK with acetylcholine receptor complexes. Grip, another PDZ domaincontaining protein, is also present at the post-synaptic membrane of the neuromuscular junction and interacts with CASK in vivo in skeletal muscle. Conclusions: CASK is a member of PDZ domain-containing protein scaffold at the post-synaptic membrane of the neuromuscular junction. CASK does not cluster in response to agrin-induced acetylcholine receptor clustering suggesting a novel mechanism of localization of CASK to the neuromuscular junction.