60 results on '"Markus A. Rüegg"'
Search Results
2. Neuronal LRP4 regulates synapse formation in the developing CNS
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Georgios Kalamakis, Carmen Ruiz de Almodovar, Karl-Klaus Conzelmann, Stephan Kröger, Anna Schick, Benedikt Berninger, Nicolás Marichal, Andromachi Karakatsani, Severino Urban, Markus A. Rüegg, Alexander Ghanem, Yina Zhang, and Susan Gascon
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0301 basic medicine ,Dendritic spine ,Rabies ,Synaptogenesis ,Hippocampus ,Biology ,Hippocampal formation ,Neuromuscular junction ,Gene Knockout Techniques ,Mice ,03 medical and health sciences ,0302 clinical medicine ,medicine ,Animals ,Lrp4 ,Central Nervous System Development ,Synapse Formation ,Dendritogenesis ,Transsynaptic Tracing ,Agrin ,In Utero Electroporation ,Psd95 ,Bassoon ,Mouse ,Molecular Biology ,Cells, Cultured ,LDL-Receptor Related Proteins ,Cerebral Cortex ,Gene knockdown ,Dendrites ,Cortex (botany) ,Cell biology ,Mice, Inbred C57BL ,030104 developmental biology ,medicine.anatomical_structure ,Receptors, LDL ,nervous system ,Rabies virus ,Synapses ,Immunology ,030217 neurology & neurosurgery ,Developmental Biology - Abstract
The low-density lipoprotein receptor-related protein 4 (LRP4) is essential in muscle fibers for the establishment of the neuromuscular junction. Here, we show that LRP4 is also expressed by embryonic cortical and hippocampal neurons, and that downregulation of LRP4 in these neurons causes a reduction in density of synapses and number of primary dendrites. Accordingly, overexpression of LRP4 in cultured neurons had the opposite effect inducing more but shorter primary dendrites with an increased number of spines. Transsynaptic tracing mediated by rabies virus revealed a reduced number of neurons presynaptic to the cortical neurons in which LRP4 was knocked down. Moreover, neuron-specific knockdown of LRP4 by in utero electroporation of LRP4 miRNA in vivo also resulted in neurons with fewer primary dendrites and a lower density of spines in the developing cortex and hippocampus. Collectively, our results demonstrate an essential and novel role of neuronal LRP4 in dendritic development and synaptogenesis in the CNS.
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- 2017
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3. The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions
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Shuo Lin, Britta Engelhardt, Markus A. Rüegg, Gaby Enzmann, Ruth Lyck, Esther Steiner, and Stephan Kröger
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Basement membrane ,Histology ,Beta-catenin ,animal structures ,610 Medicine & health ,Occludin ,Blood–brain barrier ,Permeability ,Pathology and Forensic Medicine ,Adherens junction ,Mice ,Antigens, CD ,medicine ,Animals ,Humans ,Agrin ,Tight junctions ,beta Catenin ,Cell Proliferation ,biology ,Tight junction ,Staining and Labeling ,Cadherin ,Protein Stability ,Endothelial Cells ,Regular Article ,Cell Biology ,Adherens Junctions ,Cadherins ,Cell biology ,Protein Transport ,medicine.anatomical_structure ,HEK293 Cells ,nervous system ,Blood-Brain Barrier ,Microvessels ,biology.protein ,Zonula Occludens-1 Protein ,570 Life sciences ,Chickens ,Heparan Sulfate Proteoglycans - Abstract
Barrier characteristics of brain endothelial cells forming the blood–brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, β-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and β-catenin and the junctional protein ZO-1 to brain endothelial junctions. Electronic supplementary material The online version of this article (doi:10.1007/s00441-014-1969-7) contains supplementary material, which is available to authorized users.
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- 2014
4. Loss of astrocyte polarization upon transient focal brain ischemia as a possible mechanism to counteract early edema formation
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Benoît Zuber, Markus A. Rüegg, Melanie-Jane Hannocks, Lydia Sorokin, Shuo Lin, Gaby Enzmann, Esther Steiner, Sharang Ghavampour, and Britta Engelhardt
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Ischemia ,Mice, Transgenic ,Basement Membrane ,Brain Ischemia ,Brain ischemia ,Mice ,03 medical and health sciences ,Cellular and Molecular Neuroscience ,0302 clinical medicine ,Laminin ,Edema ,medicine ,Animals ,Potassium Channels, Inwardly Rectifying ,Neuroinflammation ,030304 developmental biology ,Aquaporin 4 ,0303 health sciences ,Agrin ,biology ,Penumbra ,Cell Polarity ,medicine.disease ,Cell biology ,medicine.anatomical_structure ,Neurology ,Blood-Brain Barrier ,Astrocytes ,biology.protein ,medicine.symptom ,Neuroscience ,030217 neurology & neurosurgery ,Astrocyte - Abstract
Brain edema is the main cause of death from brain infarction. The polarized expression of the water channel protein aquaporin-4 (AQP4) on astroglial endfeet surrounding brain microvessels suggests a role in brain water balance. Loss of astrocyte foot process anchoring to the basement membrane (BM) accompanied by the loss of polarized localization of AQP4 to astrocytic endfeet has been shown to be associated with vasogenic/extracellular edema in neuroinflammation. Here, we asked if loss of astrocyte polarity is also observed in cytotoxic/intracellular edema following focal brain ischemia after transient middle cerebral artery occlusion (tMCAO). Upon mild focal brain ischemia, we observed diminished immunostaining for the BM components laminin α4, laminin α2, and the proteoglycan agrin, in the core of the lesion, but not in BMs in the surrounding penumbra. Staining for the astrocyte endfoot anchorage protein β-dystroglycan (DG) was dramatically reduced in both the lesion core and the penumbra, and AQP4 and Kir4.1 showed a loss of polarized localization to astrocytic endfeet. Interestingly, we observed that mice deficient for agrin expression in the brain lack polarized localization of β-DG and AQP4 at astrocytic endfeet and do not develop early cytotoxic/intracellular edema following tMCAO. Taken together, these data indicate that the binding of DG to agrin embedded in the subjacent BM promotes polarized localization of AQP4 to astrocyte endfeet. Reduced DG protein levels and redistribution of AQP4 as observed upon tMCAO might therefore counteract early edema formation and reflect a beneficial mechanism operating in the brain to minimize damage upon ischemia.
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- 2012
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5. MuSK levels differ between adult skeletal muscles and influence postsynaptic plasticity
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Shuo Lin, Sarina Meinen, Marcin Maj, Anna Rostedt Punga, and Markus A. Rüegg
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0303 health sciences ,medicine.medical_specialty ,animal structures ,Agrin ,General Neuroscience ,Biology ,Congenital myasthenic syndrome ,musculoskeletal system ,medicine.disease ,Myasthenia gravis ,Neuromuscular junction ,03 medical and health sciences ,0302 clinical medicine ,Endocrinology ,medicine.anatomical_structure ,Postsynaptic potential ,Internal medicine ,medicine ,Receptor ,030217 neurology & neurosurgery ,030304 developmental biology ,Acetylcholine receptor ,G alpha subunit - Abstract
Muscle-specific tyrosine kinase (MuSK) is involved in the formation and maintenance of the neuromuscular junction (NMJ), and is necessary for NMJ integrity. As muscle involvement is strikingly selective in pathological conditions in which MuSK is targeted, including congenital myasthenic syndrome with MuSK mutation and MuSK antibody-seropositive myasthenia gravis, we hypothesized that the postsynaptic response to MuSK-agrin signalling differs between adult muscles. Transcript levels of postsynaptic proteins were compared between different muscles in wild-type adult mice. MuSK expression was high in the soleus and sternomastoid muscles and low in the extensor digitorum longus (EDL) and omohyoid muscles. The acetylcholine receptor (AChR) alpha subunit followed a similar expression pattern, whereas expression of Dok-7, Lrp4 and rapsyn was comparable between the muscles. We subsequently examined muscles in mice that overexpressed a miniaturized form of neural agrin or MuSK. In these transgenic mice, the soleus and sternomastoid muscles responded with formation of ectopic AChR clusters, whereas such clusters were almost absent in the EDL and omohyoid muscles. Electroporation of Dok-7 revealed its important role as an activator of MuSK in AChR cluster formation in adult muscles. Together, our findings indicate for the first time that adult skeletal muscles harbour different endogenous levels of MuSK and that these levels determine the ability to form ectopic AChR clusters upon overexpression of agrin or MuSK. We believe that these findings are important for our understanding of adult muscle plasticity and the selective muscle involvement in neuromuscular disorders in which MuSK is diminished.
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- 2011
6. Apoptosis inhibitors and mini-agrin have additive benefits in congenital muscular dystrophy mice
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Raphael Thurnherr, Shuo Lin, Markus A. Rüegg, Sarina Meinen, Thomas Meier, and Michael Erb
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Programmed cell death ,animal structures ,Transgene ,extracellular matrix ,Mice, Transgenic ,Biology ,Muscular Dystrophies ,Extracellular matrix ,Rodent Diseases ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Fibrosis ,Omigapil ,medicine ,merosin ,Animals ,Agrin ,030304 developmental biology ,0303 health sciences ,muscle regeneration ,Histocytochemistry ,Muscles ,medicine.disease ,Immunohistochemistry ,Survival Analysis ,3. Good health ,Cell biology ,Disease Models, Animal ,cell death ,nervous system ,Neuromuscular Agents ,Proto-Oncogene Proteins c-bcl-2 ,Apoptosis ,Immunology ,Oxepins ,Congenital muscular dystrophy ,Molecular Medicine ,Laminin ,030217 neurology & neurosurgery ,medicine.drug ,Research Article ,laminin-211 - Abstract
Mutations in LAMA2 cause a severe form of congenital muscular dystrophy, called MDC1A. Studies in mouse models have shown that transgenic expression of a designed, miniaturized form of the extracellular matrix molecule agrin ('mini-agrin') or apoptosis inhibition by either overexpression of Bcl2 or application of the pharmacological substance omigapil can ameliorate the disease. Here, we tested whether mini-agrin and anti-apoptotic agents act on different pathways and thus exert additive benefits in MDC1A mouse models. By combining mini-agrin with either transgenic Bcl2 expression or oral omigapil application, we show that the ameliorating effect of mini-agrin, which acts by restoring the mechanical stability of muscle fibres and, thereby, reduces muscle fibre breakdown and concomitant fibrosis, is complemented by apoptosis inhibitors, which prevent the loss of muscle fibres. Treatment of mice with both agents results in improved muscle regeneration and increased force. Our results show that the combination of mini-agrin and anti-apoptosis treatment has beneficial effects that are significantly bigger than the individual treatments and suggest that such a strategy might also be applicable to MDC1A patients.
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- 2011
7. Identification of an Agrin Mutation that Causes Congenital Myasthenia and Affects Synapse Function
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Michel Fardeau, Laurent Schaeffer, Thierry Kuntzer, Isabelle Grosjean, Daniel Hantaï, A. Rouche, Annie Chaboud, Asma Ben Ammar, Nektaria Alexandri, Frédéric Chevessier, Caroline Huzé, Emmanuel Fournier, Andrea Brancaccio, K. Gaudon, Bruno Eymard, Jeanine Koenig, S. Bauche, Evelyne Goillot, Véronique Bernard, Heba-Aude Lecuyer, Pascale Richard, Markus A. Rüegg, Laboratoire de Biologie Moléculaire de la Cellule ( LBMC ), École normale supérieure - Lyon ( ENS Lyon ) -Université Claude Bernard Lyon 1 ( UCBL ), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Centre de Recherche de l'Institut du Cerveau et de la Moelle épinière ( CRICM ), Université Pierre et Marie Curie - Paris 6 ( UPMC ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Unité Fonctionnelle de Cardiogénétique et Myogénétique Moléculaire et Cellulaire, Assistance publique - Hôpitaux de Paris (AP-HP)-Centre de Génétique Moléculaire et Chromosomique du GH Pitié-Salpêtrière-CHU Pitié-Salpêtrière [APHP], Max-Planck-Institut für Medizinische Forschung, Max-Planck-Gesellschaft, Institut National de Neurologie, Université Tunis El Manar ( UTM ), Plateau d'analyse des protéines, IFR128, Biologie des Jonctions Neuromusculaires Normales et Pathologiques ( U686 ), Université Paris Descartes - Paris 5 ( UPD5 ) -Institut National de la Santé et de la Recherche Médicale ( INSERM ) -Centre National de la Recherche Scientifique ( CNRS ), Nerve-muscle unit, neurology service, Université de Lausanne ( UNIL ) -Centre Hospitalier Universitaire Vaudois [Lausanne] ( CHUV ), Centre de référence des maladies rares neuromusculaires, Assistance publique - Hôpitaux de Paris (AP-HP)-CHU Pitié-Salpêtrière [APHP], Service d'électrophysiologie, Istituto di Chimica del Riconoscimento Molecolare, Università Cattolica del Sacro Cuore, Biozentrum, University of Basel ( Unibas ), ANR-07-MRAR-0001,MRAR,Syndromes myasthéniques congénitaux : le réseau français et approches fondamentales ( 2007 ), Hantaï, Daniel, Programme Pluriannuel de Recherche sur les Maladies Rares (MRAR) - Implication de CXCL13 et CCL21 dans les mécanismes pathogéniques de la Myasthenie. - - MG chemokines2006 - ANR-06-MRAR-0001 - MRAR - VALID, Laboratoire de Biologie Moléculaire de la Cellule (LBMC), École normale supérieure - Lyon (ENS Lyon)-Université Claude Bernard Lyon 1 (UCBL), Université de Lyon-Université de Lyon-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Centre de Recherche de l'Institut du Cerveau et de la Moelle épinière (CRICM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Sorbonne Université (SU), Université de Tunis El Manar (UTM), Biologie des Jonctions Neuromusculaires Normales et Pathologiques (U686), Université Paris Descartes - Paris 5 (UPD5)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Neuroscience Paris Seine (NPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS), Université de Lausanne = University of Lausanne (UNIL)-Centre Hospitalier Universitaire Vaudois [Lausanne] (CHUV), Centre de référence des maladies rares neuromusculaires [CHU Pitié-Salpétriêre], CHU Pitié-Salpêtrière [AP-HP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Università cattolica del Sacro Cuore [Milano] (Unicatt), Biozentrum [Basel, Suisse], University of Basel (Unibas), ANR-06-MRAR-0001,MG chemokines,Implication de CXCL13 et CCL21 dans les mécanismes pathogéniques de la Myasthenie.(2006), École normale supérieure de Lyon (ENS de Lyon)-Université Claude Bernard Lyon 1 (UCBL), Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Université Pierre et Marie Curie - Paris 6 (UPMC), Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université (SU), Neurosciences Paris Seine (NPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Biologie Paris Seine (IBPS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Centre Hospitalier Universitaire Vaudois [Lausanne] (CHUV)-Université de Lausanne (UNIL), Sorbonne Université (SU)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Biologie Paris Seine (IBPS), Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Sorbonne Université (SU)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Institut du Cerveau et de la Moëlle Epinière = Brain and Spine Institute (ICM), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Centre National de la Recherche Scientifique (CNRS)-CHU Pitié-Salpêtrière [AP-HP], Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université, Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Institut National de la Santé et de la Recherche Médicale (INSERM)-Sorbonne Université (SU)-Centre National de la Recherche Scientifique (CNRS)-Centre National de la Recherche Scientifique (CNRS), Université Pierre et Marie Curie - Paris 6 (UPMC)-Institut National de la Santé et de la Recherche Médicale (INSERM)-CHU Pitié-Salpêtrière [AP-HP], and Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Sorbonne Université-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Centre National de la Recherche Scientifique (CNRS)
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Male ,Biopsy ,DNA Mutational Analysis ,Muscle Fibers, Skeletal ,Mutant ,[SDV.GEN] Life Sciences [q-bio]/Genetics ,0302 clinical medicine ,Mutant protein ,CHRNE ,Receptors, Cholinergic ,Genetics(clinical) ,Dystroglycans ,ComputingMilieux_MISCELLANEOUS ,Genetics (clinical) ,Genetics ,0303 health sciences ,Agrin ,Congenital myasthenic syndrome ,Recombinant Proteins ,Pedigree ,Cell biology ,medicine.anatomical_structure ,Female ,[SDV.NEU]Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC] ,Dok-7 ,Adult ,animal structures ,Mutation, Missense ,Neuromuscular Junction ,Biology ,Article ,Neuromuscular junction ,Cell Line ,03 medical and health sciences ,medicine ,Animals ,Humans ,[SDV.NEU] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC] ,Muscle, Skeletal ,030304 developmental biology ,Acetylcholine receptor ,Myasthenic Syndromes, Congenital ,[SDV.GEN]Life Sciences [q-bio]/Genetics ,Correction ,medicine.disease ,Protein Structure, Tertiary ,Rats ,Models, Chemical ,[ SDV.NEU ] Life Sciences [q-bio]/Neurons and Cognition [q-bio.NC] ,Synapses ,030221 ophthalmology & optometry ,biology.protein ,Agrin/chemistry ,Agrin/genetics ,Dystroglycans/metabolism ,Muscle Fibers, Skeletal/cytology ,Muscle Fibers, Skeletal/metabolism ,Muscle, Skeletal/metabolism ,Muscle, Skeletal/pathology ,Myasthenic Syndromes, Congenital/genetics ,Neuromuscular Junction/genetics ,Neuromuscular Junction/metabolism ,Receptors, Cholinergic/genetics ,Receptors, Cholinergic/metabolism ,Recombinant Proteins/chemistry ,Recombinant Proteins/metabolism ,Synapses/metabolism ,[ SDV.GEN ] Life Sciences [q-bio]/Genetics ,030217 neurology & neurosurgery - Abstract
International audience; We report the case of a congenital myasthenic syndrome due to a mutation in AGRN, the gene encoding agrin, an extracellular matrix molecule released by the nerve and critical for formation of the neuromuscular junction. Gene analysis identified a homozygous missense mutation, c.5125G>C, leading to the p.Gly1709Arg variant. The muscle-biopsy specimen showed a major disorganization of the neuromuscular junction, including changes in the nerve-terminal cytoskeleton and fragmentation of the synaptic gutters. Experiments performed in nonmuscle cells or in cultured C2C12 myotubes and using recombinant mini-agrin for the mutated and the wild-type forms showed that the mutated form did not impair the activation of MuSK or change the total number of induced acetylcholine receptor aggregates. A solid-phase assay using the dystrophin glycoprotein complex showed that the mutation did not affect the binding of agrin to alpha-dystroglycan. Injection of wild-type or mutated agrin into rat soleus muscle induced the formation of nonsynaptic acetylcholine receptor clusters, but the mutant protein specifically destabilized the endogenous neuromuscular junctions. Importantly, the changes observed in rat muscle injected with mutant agrin recapitulated the pre- and post-synaptic modifications observed in the patient. These results indicate that the mutation does not interfere with the ability of agrin to induce postsynaptic structures but that it dramatically perturbs the maintenance of the neuromuscular junction.
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- 2009
8. Conjugation of LG Domains of Agrins and Perlecan to Polymerizing Laminin-2 Promotes Acetylcholine Receptor Clustering
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Peter D. Yurchenco, Patrizia Barzaghi, Karen K. McKee, Sergei P. Smirnov, and Markus A. Rüegg
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DNA, Complementary ,Time Factors ,animal structures ,Polymers ,Recombinant Fusion Proteins ,Muscle Fibers, Skeletal ,Neuromuscular Junction ,Perlecan ,Models, Biological ,Biochemistry ,Basement Membrane ,Neuromuscular junction ,Mice ,chemistry.chemical_compound ,Laminin ,medicine ,Animals ,Cluster Analysis ,Receptors, Cholinergic ,Agrin ,Phosphorylation ,Molecular Biology ,Acetylcholine receptor ,Neurons ,Basement membrane ,Muscle Cells ,Membrane Glycoproteins ,Dose-Response Relationship, Drug ,biology ,Chemistry ,Myogenesis ,Cell Membrane ,Tyrosine phosphorylation ,Cell Biology ,musculoskeletal system ,Protein Structure, Tertiary ,Cell biology ,medicine.anatomical_structure ,Genetic Techniques ,nervous system ,Synapses ,biology.protein ,Tyrosine ,Chickens ,Heparan Sulfate Proteoglycans ,Protein Binding - Abstract
Neuromuscular junction (NMJ) assembly is characterized by the clustering and neuronal alignment of acetylcholine receptors (AChRs). In this study we have addressed post-synaptic contributions to assembly that may arise from the NMJ basement membrane with cultured myotubes. We show that the cell surface-binding LG domains of non-neural (muscle) agrin and perlecan promote AChR clustering in the presence of laminin-2. This type of AChR clustering occurs with a several hour lag, requires muscle-specific kinase (MuSK), and is accompanied by tyrosine phosphorylation of MuSK and betaAChR. It also requires conjugation of the agrin or perlecan to laminin together with laminin polymerization. Furthermore, AChR clustering can be mimicked with antibody binding to non-neural agrin, supporting a mechanism of ligand aggregation. Neural agrin, in addition to its unique ability to cluster AChRs through its B/z sequence insert, also exhibits laminin-dependent AChR clustering, the latter enhancing and stabilizing its activity. Finally, we show that type IV collagen, which lacks clustering activity on its own, stabilizes laminin-dependent AChR clusters. These findings provide evidence for cooperative and partially redundant MuSK-dependent functions of basement membrane in AChR assembly that can enhance neural agrin activity yet operate in its absence. Such interactions may contribute to the assembly of aneural AChR clusters that precede neural agrin release as well as affect later NMJ development.
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- 2005
9. Structure and laminin-binding specificity of the NtA domain expressed in eukaryotic cells
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Joseph B. Mascarenhas, Johannes A. Eble, Markus A. Rüegg, Takako Sasaki, Jörg Stetefeld, and Jürgen Engel
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Models, Molecular ,Protein Conformation ,Neuromuscular Junction ,Protein Structure, Secondary ,Cell Line ,law.invention ,Mice ,Protein structure ,Laminin ,law ,Escherichia coli ,Animals ,Humans ,Protein Isoforms ,Agrin ,Cysteine ,Disulfides ,Laminin binding ,Molecular Biology ,chemistry.chemical_classification ,Dose-Response Relationship, Drug ,biology ,HEK 293 cells ,Cell Differentiation ,Recombinant Proteins ,Protein Structure, Tertiary ,Rats ,Amino acid ,Biochemistry ,chemistry ,biology.protein ,Recombinant DNA ,Chickens - Abstract
Agrin is a key organizer for postsynaptic differentiation at the neuromuscular junction (NMJ). This activity requires the binding of agrin to the synaptic basal lamina via its N-terminal (NtA) domain. It has been suggested that this binding is mediated by conserved amino acids in the gamma 1 chain of laminin. Here, we report the crystal structure of chicken NtA expressed in eukaryotic HEK293 cells. In contrast to the previously published structure [Stetefeld, J., Jenny, M., Schulthess, T., Landwehr, R., Schumacher, B., Frank, S., Ruegg, M.A., Engel, J., Kammerer, R.A., 2001. The laminin-binding domain of agrin is structurally related to N-TIMP-1. Nat. Struct. Biol., 8, 705-709.], which was derived from the NtA domain expressed in E. coli, the new data show that the N-terminal tail region (amino acid residues Asn1-Arg5) is highly structured. Moreover, the disulfide bridge between Cys2 and Cys74 was also present. In addition, we show that the binding of NtA requires the gamma 1 chain of laminin and is not greatly affected by the composition of beta chains. These results confirm a model of the NtA-laminin complex where conserved amino acids in the gamma 1 chain are prerequisite for the binding to agrin and they further emphasize that the source of protein can be critical in structure determination.
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- 2005
10. Modulation of Agrin Function by Alternative Splicing and Ca2+ Binding
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Richard A. Kammerer, Ruth Landwehr, Markus A. Rüegg, Sabine Frank, Mark W. Maciejewski, Klara Rathgeb-Szabo, Andrei T. Alexandrescu, Therese Schulthess, Jörg Stetefeld, and Margrit Jenny
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Beta-sandwich ,animal structures ,Molecular Sequence Data ,Crystallography, X-Ray ,Neuromuscular junction ,03 medical and health sciences ,Mice ,0302 clinical medicine ,Postsynaptic potential ,Structural Biology ,medicine ,Animals ,Humans ,splice ,Agrin ,Amino Acid Sequence ,Molecular Biology ,030304 developmental biology ,Acetylcholine receptor ,0303 health sciences ,Binding Sites ,biology ,Chemistry ,Alternative splicing ,Protein Structure, Tertiary ,Rats ,Crystallography ,Alternative Splicing ,medicine.anatomical_structure ,nervous system ,Proteoglycan ,biology.protein ,Biophysics ,Calcium ,Sequence Alignment ,030217 neurology & neurosurgery - Abstract
The aggregation of acetylcholine receptors on postsynaptic membranes is a key step in neuromuscular junction development. This process depends on alternatively spliced forms of the proteoglycan agrin with “B-inserts” of 8, 11, or 19 residues in the protein's globular C-terminal domain, G3. Structures of the neural B8 and B11 forms of agrin-G3 were determined by X-ray crystallography. The structure of G3-B0, which lacks inserts, was determined by NMR. The agrin-G3 domain adopts a β jellyroll fold. The B insert site is flanked by four loops on one edge of the β sandwich. The loops form a surface that corresponds to a versatile interaction interface in the family of structurally related LNS proteins. NMR and X-ray data indicate that this interaction interface is flexible in agrin-G3 and that flexibility is reduced by Ca2+ binding. The plasticity of the interaction interface could enable different splice forms of agrin to select between multiple binding partners.
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- 2004
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11. Mapping of the laminin-binding site of the N-terminal agrin domain (NtA)
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Uwe Winzen, Willi Halfter, Jürgen Engel, Markus A. Rüegg, Jörg Stetefeld, and Joseph B. Mascarenhas
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Models, Molecular ,animal structures ,Molecular Sequence Data ,Neuromuscular Junction ,Protein Structure, Secondary ,General Biochemistry, Genetics and Molecular Biology ,Neuromuscular junction ,Cell Line ,Epitopes ,Laminin ,medicine ,Animals ,Humans ,Agrin ,Amino Acid Sequence ,Laminin binding ,Molecular Biology ,Acetylcholine receptor ,Binding Sites ,General Immunology and Microbiology ,biology ,General Neuroscience ,Articles ,Protein Structure, Tertiary ,Heptad repeat ,medicine.anatomical_structure ,Biochemistry ,Helix ,Mutagenesis, Site-Directed ,biology.protein ,Biophysics ,Basal lamina ,Sequence Alignment ,Protein Binding - Abstract
Agrin is a key organizer of acetylcholine receptor (AChR) clustering at the neuromuscular junction. The binding of agrin to laminin is required for its localization to synaptic basal lamina and other basement membranes. The high-affinity interaction with the coiled-coil domain of laminin is mediated by the N-terminal domain of agrin. We have adopted a structurally guided site-directed mutagenesis approach to map the laminin-binding site of NtA. Mutations of L117 and V124 in the C-terminal helix 3 showed that they are crucial for binding. Both residues are located in helix 3 and face the groove between the beta-barrel and the C-terminal helical segment of NtA. Remarkably, the distance between both residues matches a heptad repeat distance of two aliphatic residues which are solvent exposed in the coiled-coil domain of laminin. A lower but significant contribution originates from R43 and a charged cluster (E23, E24 and R40) at the open face of the beta-barrel structure. We propose that surface-exposed, conserved residues of the laminin gamma1 chain interact with NtA via hydrophobic and ionic interactions.
- Published
- 2003
12. An Intrinsic Distinction in Neuromuscular Junction Assembly and Maintenance in Different Skeletal Muscles
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Markus Sigrist, Thomas M. Jessell, Markus A. Rüegg, Alexandre Ferrao Santos, Joshua R. Sanes, San Pun, Silvia Arber, and Pico Caroni
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animal structures ,Neuroscience(all) ,Models, Neurological ,Neuromuscular Junction ,Biology ,Neuromuscular junction ,Mice ,Postsynaptic potential ,medicine ,Paralysis ,Animals ,Receptors, Cholinergic ,Agrin ,Botulinum Toxins, Type A ,Muscle, Skeletal ,Acetylcholine receptor ,Mice, Knockout ,Denervation ,General Neuroscience ,Receptor Aggregation ,Embryo ,Bungarotoxins ,Immunohistochemistry ,Muscle Denervation ,Hindlimb ,medicine.anatomical_structure ,Neuromuscular Agents ,nervous system ,Schwann Cells ,medicine.symptom ,Neuroscience - Abstract
We analyzed the formation of neuromuscular junctions (NMJs) in individual muscles of the mouse embryo. Skeletal muscles can be assigned to one of two distinct classes of muscles, termed “Fast Synapsing” (FaSyn) and “Delayed Synapsing” (DeSyn) muscles, which differ significantly with respect to the initial focal clustering of postsynaptic AChRs, the timing of presynaptic maturation, and the maintenance of NMJs in young adult mice. Differences between classes were intrinsic to the muscles and manifested in the absence of innervation or agrin. Paralysis or denervation of young adult muscles resulted in disassembly of AChR clusters on DeSyn muscles, whereas those on FaSyn muscles were preserved. Our results show that postsynaptic differentiation processes intrinsic to FaSyn and DeSyn muscles influence the formation of NMJs during development and their maintenance in the adult.
- Published
- 2002
- Full Text
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13. A neuronal inhibitory domain in the N-terminal half of agrin
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Fritz G. Rathjen, Cong Wang, Markus A. Rüegg, Kristine Baerwald-De La Torre, and John L. Bixby
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animal structures ,Agrin ,biology ,Neurite ,Cell adhesion molecule ,General Neuroscience ,Ciliary ganglion ,Inhibitory postsynaptic potential ,Cell biology ,Cellular and Molecular Neuroscience ,nervous system ,Laminin ,Postsynaptic potential ,biology.protein ,Immunoglobulin superfamily - Abstract
Agrin is required for appropriate pre- and postsynaptic differentiation of neuromuscular junctions. While agrin's ability to orchestrate postsynaptic differentiation is well documented, more recent experiments have suggested that agrin is also a "stop signal" for the presynaptic neuron, and that agrin has actions on neurons in the CNS. To elucidate the neuronal activities of agrin and to define the receptor(s) responsible for these functions, we have examined adhesions of neurons and their neurite-outgrowth responses to purified agrin in vitro. We find that both full-length agrin and the C-terminal 95 kDa of agrin (agrin c95), which is sufficient to induce postsynaptic differentiation, are adhesive for chick ciliary ganglion (CG) and forebrain neurons. Consistent with previous findings, our results show that N-CAM binds to full-length agrin, and suggest that alpha-dystroglycan is a neuronal receptor for agrin c95. In neurite outgrowth assays, full-length agrin inhibited both laminin- and N-cadherin-induced neurite growth from CG neurons. The N-terminal 150 kDa fragment of agrin, but not agrin c95, inhibited neurite outgrowth, indicating that domains in the N-terminal portion of agrin are sufficient for this function. Adhesion assays using protein-coated beads and agrin-expressing cells revealed differential interactions of agrin with members of the immunoglobulin superfamily of cell adhesion molecules. However, none of these, including N-CAM, appeared to be critical for neuronal adhesion. In summary, our results suggest that the N-terminal half of agrin is involved in agrin's ability to inhibit neurite outgrowth. Our results further suggest that neither alpha-dystroglycan nor N-CAM, two known binding proteins for agrin, mediate this effect.
- Published
- 2002
14. Differential regulation of AChR clustering in the polar and equatorial region of murine muscle spindles
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Stephan Kröger, Andromachi Karakatsani, Yina Zhang, Shuo Lin, and Markus A. Rüegg
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Receptor complex ,animal structures ,Sensory Receptor Cells ,Synaptogenesis ,Neuromuscular Junction ,Mice, Transgenic ,Biology ,Receptors, Nicotinic ,Neuromuscular junction ,Bacterial Proteins ,Anterior Horn Cells ,Ganglia, Spinal ,medicine ,Animals ,Agrin ,Muscle Spindles ,LDL-Receptor Related Proteins ,Acetylcholine receptor ,Mice, Knockout ,Motor Neurons ,Lumbar Vertebrae ,Microscopy, Confocal ,General Neuroscience ,Receptor Protein-Tyrosine Kinases ,musculoskeletal system ,Intrafusal muscle fiber ,Immunohistochemistry ,Sensory neuron ,Cell biology ,Nicotinic acetylcholine receptor ,Luminescent Proteins ,medicine.anatomical_structure ,nervous system ,Receptors, LDL ,Neuroscience - Abstract
Intrafusal fibers of muscle spindles are innervated in the central region by afferent sensory axons and at both polar regions by efferent γ-motoneurons. We previously demonstrated that both neuron-muscle contact sites contain cholinergic synapse-like specialisation, including aggregates of the nicotinic acetylcholine receptor (AChR). In this study we tested the hypothesis that agrin and its receptor complex (consisting of LRP4 and the tyrosine kinase MuSK) are involved in the aggregation of AChRs in muscle spindles, similar to their role at the neuromuscular junction. We show that agrin, MuSK and LRP4 are concentrated at the contact site between the intrafusal fibers and the sensory- and γ-motoneuron, respectively, and that they are expressed in the cell bodies of proprioceptive neurons in dorsal root ganglia. Moreover, agrin and LRP4, but not MuSK, are expressed in γ-motoneuron cell bodies in the ventral horn of the spinal cord. In agrin- and in MuSK-deficient mice, AChR aggregates are absent from the polar regions. In contrast, the subcellular concentration of AChRs in the central region where the sensory neuron contacts the intrafusal muscle fiber is apparently unaffected. Skeletal muscle-specific expression of miniagrin in agrin(-/-) mice in vivo is sufficient to restore the formation of γ-motoneuron endplates. These results show that agrin and MuSK are major determinants during the formation of γ-motoneuron endplates but appear dispensable for the aggregation of AChRs at the central region. Our results therefore suggest different molecular mechanisms for AChR clustering within two domains of intrafusal fibers.
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- 2014
15. Injection of a soluble fragment of neural agrin (NT-1654) considerably improves the muscle pathology caused by the disassembly of the neuromuscular junction
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Shuo Lin, Monika Haubitz, Filippo Oliveri, Jan Wim Vrijbloed, Markus A. Rüegg, Stefan Kucsera, Stefan Hettwer, and Ruggero Fariello
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Sarcopenia ,Mouse ,Muscle Fibers, Skeletal ,lcsh:Medicine ,Biochemistry ,Muscular Dystrophies ,Extracellular matrix ,Motor Neuron Diseases ,Mice ,Drug Discovery ,Receptors, Cholinergic ,Receptor ,lcsh:Science ,Denervation ,Multidisciplinary ,Agrin ,Serine Endopeptidases ,Anatomy ,Animal Models ,Neuromuscular Diseases ,Sciatic Nerve ,Cell biology ,Slow-Twitch Muscle Fiber ,medicine.anatomical_structure ,Phenotype ,Neurology ,Medicine ,Sciatic nerve ,Research Article ,animal structures ,Nerve Crush ,Neuromuscular Junction ,Biology ,Signaling Pathways ,Neuromuscular junction ,Injections ,Model Organisms ,medicine ,Animals ,Humans ,Muscle Strength ,Muscle, Skeletal ,Motor Systems ,lcsh:R ,Body Weight ,medicine.disease ,Mice, Inbred C57BL ,HEK293 Cells ,Solubility ,nervous system ,lcsh:Q ,Molecular Neuroscience ,Neuroscience - Abstract
Treatment of neuromuscular diseases is still an unsolved problem. Evidence over the last years strongly indicates the involvement of malformation and dysfunction of neuromuscular junctions in the development of such medical conditions. Stabilization of NMJs thus seems to be a promising approach to attenuate the disease progression of muscle wasting diseases. An important pathway for the formation and maintenance of NMJs is the agrin/Lrp4/MuSK pathway. Here we demonstrate that the agrin biologic NT-1654 is capable of activating the agrin/Lrp4/MuSK system in vivo, leading to an almost full reversal of the sarcopenia-like phenotype in neurotrypsin-overexpressing (SARCO) mice. We also show that injection of NT-1654 accelerates muscle re-innervation after nerve crush. This report demonstrates that a systemically administered agrin fragment has the potential to counteract the symptoms of neuromuscular disorders.
- Published
- 2014
16. [Untitled]
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Markus A. Rüegg, Margrit Jenny, Jürgen Engel, Jörg Stetefeld, Ruth Landwehr, Sabine Frank, Therese Schulthess, Richard A. Kammerer, and Beat Schumacher
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Coiled coil ,animal structures ,Agrin ,Structural similarity ,Plasma protein binding ,Biology ,Biochemistry ,Neuromuscular junction ,Cell biology ,Protein structure ,medicine.anatomical_structure ,nervous system ,Structural Biology ,Genetics ,medicine ,Binding site ,Laminin binding - Abstract
Agrin is the key organizer of postsynaptic differentiation at the neuromuscular junction. This organization activity requires the binding of agrin to the synaptic basal lamina. Binding is conferred by the N-terminal agrin (NtA) domain, which mediates a high-affinity interaction with the coiled coil domain of laminins. Here, we report the crystal structure of chicken NtA at 1.6 A resolution. The structure reveals that NtA harbors an oligosaccharide/oligonucleotide-binding fold with several possible sites for the interaction with different ligands. A high structural similarity of NtA with the protease inhibition domain in tissue inhibitor of metalloproteinases-1 (TIMP-1) supports the idea of additional functions of agrin besides synaptogenic activity.
- Published
- 2001
17. A minigene of neural agrin encoding the laminin-binding and acetylcholine receptor-aggregating domains is sufficient to induce postsynaptic differentiation in muscle fibres
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P A Marangi, D. M. Hauser, Markus A. Rüegg, Thomas Meier, J Moll, and H R Brenner
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animal structures ,Agrin ,biology ,General Neuroscience ,Neuromuscular junction ,Cell biology ,medicine.anatomical_structure ,nervous system ,Laminin ,Postsynaptic potential ,medicine ,biology.protein ,Myocyte ,Basal lamina ,Laminin binding ,Neuroscience ,Acetylcholine receptor - Abstract
The extracellular matrix molecule agrin is both necessary and sufficient for inducing the formation of postsynaptic specializations at the neuromuscular junction (NMJ). At the mature NMJ, agrin is stably incorporated in synaptic basal lamina. The postsynapse-inducing activity of chick agrin, as assayed by its capability of causing aggregation of acetylcholine receptors (AChRs) on cultured muscle cells, maps to a 21 kDa, C-terminal domain. Binding of chick agrin to muscle basal lamina is mediated by the laminins and maps to a 25 kDa, N-terminal fragment of agrin. Here we show that an expression construct encoding a 'mini'-agrin, in which the laminin-binding fragment was fused to the AChR-clustering domain, is sufficient to induce postsynaptic differentiation in vivo when injected into non-synaptic sites of rat soleus muscle. As shown for ectopic postsynaptic differentiation induced by full-length neural agrin, myonuclei underneath the ectopic sites expressed the gene for the AChR epsilon-subunit. Altogether, our data show that a 'mini'-agrin construct encoding only a small fraction of the entire agrin protein is sufficient to induce postsynapse-like structures that are reminiscent of those induced by full-length neural agrin or innervation by motor neurons.
- Published
- 1998
18. Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane
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Lambert P. van den Heuvel, Karel J.M. Assmann, H. Dijkman, Thea J. van de Velden, Jacques H. Veerkamp, C.A.F. Buskens, Leo A. H. Monnens, Markus A. Rüegg, Alexander J. Groffen, Jacob van den Born, Groningen Kidney Center (GKC), Groningen Institute for Organ Transplantation (GIOT), Functional Genomics, Constitutional and Administrative Law, Human genetics, and Amsterdam Neuroscience - Cellular & Molecular Mechanisms
- Subjects
0301 basic medicine ,Immunologische ontstekingsprocessen in de nier ,Kidney Glomerulus ,Pathofysiologie, immunologie en behandeling van nieraandoeningen ,Fluorescent Antibody Technique ,Immune Sera/metabolism ,Heparitin Sulfate/metabolism ,urologic and male genital diseases ,Basement Membrane ,Extracellular matrix ,Inflammatory reactions in the kidneys ,Monoclonal ,Fluorescent Antibody Technique, Indirect ,Microscopy, Immunoelectron ,Neuromuscular Junction/metabolism ,Heparan Sulfate Proteoglycans/metabolism ,Microscopy ,Agrin ,biology ,Chemistry ,Glomerular basement membrane ,Structuur en functie van heparansulfaat proteoglycanen in de humane basaal membraan van de nier ,Antibodies, Monoclonal ,Pathophysiology, immunology and treatment of renal disease ,Cell biology ,medicine.anatomical_structure ,Biochemistry ,Kidney Glomerulus/cytology ,Proteoglycans of renal basement membranes ,Muscle ,Proteoglycans ,Basal lamina ,Anatomy ,Adult ,Indirect ,Kidney Cortex ,Histology ,animal structures ,medicine.drug_class ,Immunoelectron microscopy ,Neuromuscular Junction ,Heparan sulfate proteoglycan ,Enzyme-Linked Immunosorbent Assay ,Kidney Cortex/cytology ,Perlecan ,Skeletal/metabolism ,Monoclonal antibody ,Neuromuscular junction ,Antibodies ,Fluorescence ,03 medical and health sciences ,Proteoglycanen van basaalmembranen van de nier ,Agrin/biosynthesis ,medicine ,Animals ,Humans ,Muscle, Skeletal ,Immunoelectron ,Muscle, Skeletal/metabolism ,030102 biochemistry & molecular biology ,Basement Membrane/metabolism ,Bungarotoxins/metabolism ,urogenital system ,Immune Sera ,Bungarotoxins ,Proteoglycans/metabolism ,Rats ,carbohydrates (lipids) ,030104 developmental biology ,nervous system ,Microscopy, Fluorescence ,biology.protein ,Heparitin Sulfate ,Structure and function of heparan sulphate proteoglycans in human renal basement membranes ,Heparan Sulfate Proteoglycans - Abstract
Agrin is a heparan sulfate proteoglycan (HSPG) that is highly concentrated in the synaptic basal lamina at the neuromuscular junction (NMJ). Agrin-like immunoreactivity is also detected outside the NMJ. Here we show that agrin is a major HSPG component of the human glomerular basement membrane (GBM). This is in addition to perlecan, a previously characterized HSPG of basement membranes. Antibodies against agrin and against an unidentified GBM HSPG produced a strong staining of the GBM and the NMJ, different from that observed with anti-perlecan antibodies. In addition, anti-agrin antisera recognized purified GBM HSPG and competed with an anti-GBM HSPG monoclonal antibody in ELISA. Furthermore, both antibodies recognized a molecule that migrated in SDS-PAGE as a smear and had a molecular mass of approximately 200-210 kD after deglycosylation. In immunoelectron microscopy, agrin showed a linear distribution along the GBM and was present throughout the width of the GBM. This was again different from perlecan, which was exclusively present on the endothelial side of the GBM and was distributed in a nonlinear manner. Quantitative ELISA showed that, compared with perlecan, the agrin-like GBM HSPG showed a sixfold higher molarity in crude glomerular extract. These results show that agrin is a major component of the GBM, indicating that it may play a role in renal ultrafiltration and cell matrix interaction. (J Histochem Cytochem 46:19-27, 1998)
- Published
- 1998
19. Agrin Is a High-affinity Binding Protein of Dystroglycan in Non-muscle Tissue
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Matthias Gesemann, Markus A. Rüegg, Andrea Brancaccio, Beat Schumacher, University of Zurich, and Ruegg, M A
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musculoskeletal diseases ,congenital, hereditary, and neonatal diseases and abnormalities ,1303 Biochemistry ,animal structures ,610 Medicine & health ,Kidney ,Biochemistry ,Neuromuscular junction ,1307 Cell Biology ,Dystroglycans ,Laminin ,1312 Molecular Biology ,medicine ,Dystroglycan ,Animals ,Agrin ,Muscle, Skeletal ,Lung ,Molecular Biology ,Membrane Glycoproteins ,10242 Brain Research Institute ,biology ,Chemistry ,Skeletal muscle ,Kidney metabolism ,Cell Biology ,musculoskeletal system ,Molecular biology ,Cytoskeletal Proteins ,medicine.anatomical_structure ,nervous system ,COS Cells ,biology.protein ,570 Life sciences ,Pikachurin ,Chickens ,Protein Binding - Abstract
Agrin is a basement membrane-associated proteoglycan that induces the formation of postsynaptic specializations at the neuromuscular junction. This activity is modulated by alternative splicing and is thought to be mediated by receptors expressed in muscle fibers. An isoform of agrin that does not induce postsynaptic specializations binds with high affinity to dystroglycan, a component of the dystrophin-glycoprotein complex. Transcripts encoding this agrin isoform are expressed in a variety of non-muscle tissues. Here, we analyzed the tissue distribution of agrin and dystroglycan on the protein level and determined their binding affinities. We found that agrin is most abundant in lung, kidney, and brain. Only a little agrin was detected in skeletal muscle, and no agrin was found in liver. Dystroglycan was highly expressed in all tissues examined except in liver. In a solid-phase radioligand binding assay, agrin bound to dystroglycan from lung, kidney, and skeletal muscle with a dissociation constant between 1.8 and 2.2 nM, while the affinity to brain-derived dystroglycan was 4.6 nM. In adult kidney and lung, agrin co-purified and co-immunoprecipitated with dystroglycan, and both molecules were co-localized in embryonic tissue. These data show that the agrin isoform expressed in non-muscle tissue is a high-affinity binding partner of dystroglycan and they suggest that this interaction, like that between laminin and dystroglycan, may be important for the mechanical integrity of the tissue.
- Published
- 1998
20. Neural Agrin Induces Ectopic Postsynaptic Specializations in Innervated Muscle Fibers
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Lukas Landmann, Markus A. Rüegg, Thomas Meier, Matthias Chiquet, Dominik M. Hauser, and Hans Rudolf Brenner
- Subjects
Gene isoform ,animal structures ,Receptor, ErbB-2 ,Protein subunit ,Muscle Fibers, Skeletal ,Nerve Tissue Proteins ,Motor Endplate ,Sodium Channels ,Substrate Specificity ,Postsynaptic potential ,Gene expression ,Utrophin ,Animals ,Receptors, Cholinergic ,ERBB3 ,Agrin ,Nerve Tissue ,Acetylcholine receptor ,Chemistry ,Muscles ,General Neuroscience ,Receptor Aggregation ,Receptor Protein-Tyrosine Kinases ,Articles ,Rats ,Cell biology ,Electrophysiology ,ErbB Receptors ,Synapses ,Chickens ,Neuroscience - Abstract
Neural agrin, in the absence of a nerve terminal, can induce the activity-resistant expression of acetylcholine receptor (AChR) subunit genes and the clustering of synapse-specific adult-type AChR channels in nonsynaptic regions of adult skeletal muscle fibers. Here we show that, when expression plasmids for neural agrin are injected into the extrasynaptic region of innervated muscle fibers, the following components of the postsynaptic apparatus are aggregated and colocalized with ectopic agrin-induced AChR clusters: laminin-β2, MuSK, phosphotyrosine-containing proteins, β-dystroglycan, utrophin, and rapsyn. These components have been implicated to play a role in the differentiation of neuromuscular junctions. Furthermore, ErbB2 and ErbB3, which are thought to be involved in the regulation of neurally induced AChR subunit gene expression, were colocalized with agrin-induced AChR aggregates at ectopic nerve-free sites. The postsynaptic muscle membrane also contained a high concentration of voltage-gated Na+channels as well as deep, basal lamina-containing invaginations comparable to the secondary synaptic folds of normal endplates. The ability to induce AChR aggregationin vivowas not observed in experiments with a muscle-specific agrin isoform. Thus, a motor neuron-specific agrin isoform is sufficient to induce a full ectopic postsynaptic apparatus in muscle fibers kept electrically active at their original endplate sites.
- Published
- 1997
21. Extracellular matrix of secondary lymphoid organs impacts on B-cell fate and survival
- Author
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Thomas Winkler, Julia Rolf, John F. Kearney, Elisabeth Georges-Labouesse, Zerina Lokmic, Jian Song, Xueli Zhang, Markus A. Rüegg, Arnoud Sonnenberg, Nathalie Horn, Melanie Jane Hannocks, Chuan Wu, Tim Lämmermann, Susanna Cardell, Rupert Hallmann, and Lydia Sorokin
- Subjects
Stromal cell ,Cell Survival ,T cell ,Population ,Integrin ,Biology ,Extracellular matrix ,Mice ,03 medical and health sciences ,0302 clinical medicine ,Bone Marrow ,Cell Movement ,Laminin ,medicine ,Animals ,Agrin ,education ,B cell ,030304 developmental biology ,Mice, Knockout ,B-Lymphocytes ,0303 health sciences ,education.field_of_study ,Multidisciplinary ,Integrin alpha6beta1 ,Marginal zone ,Extracellular Matrix ,Cell biology ,medicine.anatomical_structure ,PNAS Plus ,Immunology ,biology.protein ,Spleen ,030217 neurology & neurosurgery - Abstract
We describe a unique extracellular matrix (ECM) niche in the spleen, the marginal zone (MZ), characterized by the basement membrane glycoproteins, laminin α5 and agrin, that promotes formation of a specialized population of MZ B lymphocytes that respond rapidly to blood-borne antigens. Mice with reduced laminin α5 expression show reduced MZ B cells and increased numbers of newly formed (NF) transitional B cells that migrate from the bone marrow, without changes in other immune or stromal cell compartments. Transient integrin α6β1-mediated interaction of NF B cells with laminin α5 in the MZ supports the MZ B-cell population, their long-term survival, and antibody response. Data suggest that the unique 3D structure and biochemical composition of the ECM of lymphoid organs impacts on immune cell fate.
- Published
- 2013
- Full Text
- View/download PDF
22. Diverse functions of the extracellular matrix molecule agrin
- Author
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Alain J. Denzer, Matthias Gesemann, and Markus A. Rüegg
- Subjects
Basement membrane ,animal structures ,Agrin ,biology ,Chemistry ,General Neuroscience ,Neuromuscular junction ,Cell biology ,Extracellular matrix ,medicine.anatomical_structure ,nervous system ,Biochemistry ,Postsynaptic potential ,Dystroglycan ,biology.protein ,medicine ,Basal lamina ,Integral membrane protein - Abstract
The extracellular matrix is a well organized structure with profound effects on the development and the integrity of adherent tissues. Agrin is a component of many matrices, such as the basement membrane of kidney, blood capillaries and the muscle fiber basal lamina, where it is highly concentrated at the neuromuscular junction. During synapse formation agrin is believed to promote differentiation of the postsynaptic muscle fibers and the presynaptic motor neuron. This complex process is, at least in part, based on specific interactions of agrin with other matrix molecules and with membrane-associated or integral membrane proteins of the abutting cells. This review summarizes studies concerning the integration of agrin with other molecules and highlights possible functions of agrin in the synaptic basal lamina and in other matrices.
- Published
- 1996
23. Substrate-bound agrin induces expression of acetylcholine receptor epsilon-subunit gene in cultured mammalian muscle cells
- Author
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Stephan Kröger, Markus A. Rüegg, Alexander Herczeg, Marianne Lichtsteiner, Hans Rudolf Brenner, and Graham Jones
- Subjects
Gene isoform ,DNA, Complementary ,animal structures ,Transcription, Genetic ,Chick Embryo ,Biology ,Neuromuscular junction ,Substrate Specificity ,Rats, Sprague-Dawley ,Postsynaptic potential ,Gene expression ,medicine ,Animals ,Receptors, Cholinergic ,Agrin ,RNA, Messenger ,Cloning, Molecular ,Cells, Cultured ,Acetylcholine receptor ,Multidisciplinary ,Myogenesis ,Muscles ,Molecular biology ,Rats ,medicine.anatomical_structure ,nervous system ,Basal lamina ,Research Article - Abstract
Expression of the epsilon-subunit gene of the acetylcholine receptor (AChR) by myonuclei located at the neuromuscular junction is precisely regulated during development. A key role in this regulation is played by the synaptic portion of the basal lamina, a structure that is also known to contain agrin, a component responsible for the formation of postsynaptic specializations. We tested whether agrin has a function in synaptic AChR gene expression. Synaptic basal lamina from native adult muscle and recombinant agrin bound to various substrates induced in cultured rat myotubes AChR clusters that were colocalized with epsilon-subunit mRNA. Estimation of transcript levels by Northern hybridization analysis of total RNA showed a significant increase when myotubes were grown on substrate impregnated with agrin, but were unchanged when agrin was applied in the medium. The effect was independent of the receptor aggregating activity of the agrin isoform used, and agrin acted, at least in part, at the level of epsilon-subunit gene transcription. These findings are consistent with a role of agrin in the regulation of AChR subunit gene expression at the neuromuscular junction, which would depend on its binding to the synaptic basal lamina.
- Published
- 1996
24. Agrin, laminin β2 (s-laminin) and ARIA: their role in neuromuscular development
- Author
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Markus A. Rüegg
- Subjects
Agrin ,Synaptic cleft ,biology ,General Neuroscience ,Neuromuscular Junction ,Gene Expression ,Neuromuscular junction ,Extracellular matrix ,medicine.anatomical_structure ,Postsynaptic potential ,Laminin ,medicine ,biology.protein ,Animals ,Basal lamina ,Neuroscience ,Acetylcholine receptor - Abstract
Development of pre- and postsynaptic specializations at the vertebrate neuromuscular junction is affected by molecules concentrated in the extracellular matrix of the synaptic cleft. Agrin, laminin beta 2 and ARIA are the best characterized proteins known to be involved in particular aspects of synaptic differentiation. Recent advances in defining the domains of these molecules that are crucial for their synapse-organizing activity and their localization to synaptic basal lamina will help our understanding of the molecular mechanisms involved in synapse formation.
- Published
- 1996
25. M.I.3 The role of laminins in myomatrix assembly and skeletal muscle stability
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Sarina Meinen, P.G. Yurchenco, Geraldine Maier, Markus A. Rüegg, and Shuo Lin
- Subjects
Agrin ,Skeletal muscle ,Inflammation ,Perlecan ,Biology ,medicine.disease ,Cell biology ,medicine.anatomical_structure ,Neurology ,Cell surface receptor ,Pediatrics, Perinatology and Child Health ,Immunology ,medicine ,Congenital muscular dystrophy ,biology.protein ,Neurology (clinical) ,medicine.symptom ,Receptor ,Genetics (clinical) ,Function (biology) - Abstract
The basement membrane of skeletal muscle (myomatrix) consists of a complex network of highly glycosylated proteins. Important components of the myomatrix are the laminins and the collagens. During development, the myomatrix is assembled by interactions with cell surface receptors and by self-polymerization, which results in an extracellular scaffold important for skeletal muscle stability. Mutations in the α2 chain of laminin-211 impinge on myomatrix assembly and its interaction with cell surface receptors, and result in one of the most severe forms of congenital muscular dystrophy (MDC1A). Based on a detailed mechanistic understanding of the function of laminin-211, we have designed artificial linker molecules derived from agrin or perlecan with the aim to bridge the remaining myomatrix to cell surface receptors. In the conducted preclinical, proof-of-concept studies we were able to show that they can substantially ameliorate the disease phenotype in a mouse model of MDC1A. Subsequent approaches to further improve the treatment have focused on testing interventions that affect downstream pathways, such as apoptosis and inflammation, using pharmacological and genetic tools. All those treatments show additional beneficial effects on disease progression. However, semi-quantitative comparison of the treatments indicates that interventions that target early events in the disease are more efficacious than those targeting further downstream pathways. Finally, we will also report on the ongoing efforts to combine interventions that boost myomatrix assembly with those that connect it to skeletal muscle receptors. In summary, we will report on proof-of-concept studies that provide mechanistic insights and unequivocal preclinical evidence for the potential use of those therapies. As the MDC1A mice used in our studies share many hallmarks with the human disease, these preclinical data may be of high predictive value for future clinical studies.
- Published
- 2013
26. Muscle-wide secretion of a miniaturized form of neural agrin rescues focal neuromuscular innervation in agrin mutant mice
- Author
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Markus A. Rüegg, Hans Rudolf Brenner, Marcin Maj, Shuo Lin, Gabriela Bezakova, and Josef P. Magyar
- Subjects
medicine.medical_specialty ,animal structures ,Diaphragm ,Neuromuscular Junction ,Motor nerve ,Biology ,Cholinergic Agonists ,Muscle Development ,Neuromuscular junction ,Mice ,Postsynaptic potential ,Internal medicine ,medicine ,Animals ,Receptors, Cholinergic ,Agrin ,Acetylcholine receptor ,Motor Neurons ,Multidisciplinary ,Receptor Protein-Tyrosine Kinases ,Depolarization ,Biological Sciences ,Acetylcholine ,Mice, Mutant Strains ,Cell biology ,Protein Structure, Tertiary ,Endocrinology ,medicine.anatomical_structure ,nervous system ,Cholinergic ,Carbachol ,medicine.drug ,Signal Transduction - Abstract
Agrin and its receptor MuSK are required for the formation of the postsynaptic apparatus at the neuromuscular junction (NMJ). In the current model the local deposition of agrin by the nerve and the resulting local activation of MuSK are responsible for creating and maintaining the postsynaptic apparatus including clusters of acetylcholine receptors (AChRs). Concomitantly, the release of acetylcholine (ACh) and the resulting depolarization disperses those postsynaptic structures that are not apposed by the nerve and thus not stabilized by agrin-MuSK signaling. Here we show that a miniaturized form of agrin, consisting of the laminin-binding and MuSK-activating domains, is sufficient to fully restore NMJs in agrin mutant mice when expressed by developing muscle. Although miniagrin is expressed uniformly throughout muscle fibers and induces ectopic AChR clusters, the size and the number of those AChR clusters contacted by the motor nerve increase during development. We provide experimental evidence that this is due to ACh, because the AChR agonist carbachol stabilizes AChR clusters in organotypic cultures of embryonic diaphragms. In summary, our results show that agrin function in NMJ development requires only two small domains, and that this function does not depend on the local deposition of agrin at synapses. Finally, they suggest a novel local function of ACh in stabilizing postsynaptic structures.
- Published
- 2008
27. The Role of Nerve- versus Muscle-Derived Factors in Mammalian Neuromuscular Junction Formation
- Author
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Hans Rudolf Brenner, Markus A. Rüegg, Shuo Lin, and Lukas Landmann
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Male ,animal structures ,Transgene ,Diaphragm ,Muscle Fibers, Skeletal ,Synaptogenesis ,Neuromuscular Junction ,Muscle Proteins ,Mice, Transgenic ,Biology ,Muscle Development ,Models, Biological ,Neuromuscular junction ,Mice ,Imaging, Three-Dimensional ,Organ Culture Techniques ,medicine ,Animals ,Receptors, Cholinergic ,Agrin ,Receptor ,Acetylcholine receptor ,Regulation of gene expression ,Homeodomain Proteins ,Myogenesis ,General Neuroscience ,Receptor Aggregation ,Gene Expression Regulation, Developmental ,Receptor Protein-Tyrosine Kinases ,Articles ,musculoskeletal system ,Embryo, Mammalian ,medicine.anatomical_structure ,nervous system ,Female ,Neuroscience ,Transcription Factors - Abstract
Neuromuscular junctions (NMJs) normally form in the central region of developing muscle. In this process, agrin released from motor neurons has been considered to initiate the formation of synaptic acetylcholine receptor (AChR) clusters (neurocentric model). However, in muscle developing in the absence of nerves and thus of agrin, AChR clusters still form in the muscle center. This raises the possibility that the region of NMJ formation is determined by muscle-derived cues that spatially restrict the nerve to form synapses from aneural AChR clusters, e.g., by patterned expression of the agrin receptor MuSK (muscle-specific kinase) (myocentric model). Here we examine at initial stages of synaptogenesis whether the responsiveness of myotubes to agrin is spatially restricted, whether the regions of NMJ formation in wild-type muscle and of aneural AChR cluster formation in agrin-deficient animals correlate, and whether AChR cluster growth depends on the presence of agrin. We show that primary myotubes form AChR clusters in response to exogenous agrin in their central region only, a pattern that can spatially restrict NMJ formation. However, the nerve also makes synapses in regions in which aneural AChR clusters do not form, and agrin promotes synaptic cluster growth from the first stages of neuromuscular contact formation. These data indicate that aneural AChR clusters per se are not required for NMJ formation. A model is proposed that explains either the neurocentric or the myocentric mode of NMJ formation depending on a balance between the levels of MuSK expression and the availability of nerve-released agrin.
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- 2008
28. Altering Agrin Expression Influences Aβ Deposition in APP(Swe)/PS1(ex9) Transgenic Mice
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Markus A. Rüegg, Steven M. Rauch, Hira Chaudhry, John E. Donahue, Justin R. Fallon, Miles C. Miller, Robert W. Burgess, Conrad E. Johanson, and Edward G. Stopa
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Genetically modified mouse ,Agrin ,Chemistry ,Genetics ,Molecular Biology ,Biochemistry ,Deposition (chemistry) ,Biotechnology ,Cell biology - Published
- 2008
29. Tyrosine phosphatases such as SHP-2 act in a balance with Src-family kinases in stabilization of postsynaptic clusters of acetylcholine receptors
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Alain A. Camilleri, Markus A. Rüegg, Matthias Gesemann, Raffaella Willmann, Shuo Lin, Christian Fuhrer, Gayathri Sadasivam, University of Zurich, and Fuhrer, Christian
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Muscle Fibers, Skeletal ,2804 Cellular and Molecular Neuroscience ,Protein Tyrosine Phosphatase, Non-Receptor Type 11 ,Protein tyrosine phosphatase ,Synaptic Transmission ,Mice ,Postsynaptic potential ,Chlorocebus aethiops ,Receptors, Cholinergic ,Enzyme Inhibitors ,Tyrosine ,Mice, Knockout ,Agrin ,Myogenesis ,General Neuroscience ,lcsh:QP351-495 ,Intracellular Signaling Peptides and Proteins ,2800 General Neuroscience ,musculoskeletal system ,Cell biology ,src-Family Kinases ,medicine.anatomical_structure ,Biochemistry ,COS Cells ,Phosphorylation ,RNA Interference ,Research Article ,animal structures ,Neuromuscular Junction ,Synaptic Membranes ,Down-Regulation ,610 Medicine & health ,Biology ,Neuromuscular junction ,lcsh:RC321-571 ,Cellular and Molecular Neuroscience ,Organ Culture Techniques ,medicine ,Animals ,Muscle, Skeletal ,lcsh:Neurosciences. Biological psychiatry. Neuropsychiatry ,Acetylcholine receptor ,10242 Brain Research Institute ,Receptor Aggregation ,Receptor Protein-Tyrosine Kinases ,Acetylcholine ,lcsh:Neurophysiology and neuropsychology ,570 Life sciences ,biology ,Protein Tyrosine Phosphatases - Abstract
Background Development of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ), agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs). Postnatally, AChR aggregates are stabilized by molecular pathways that are poorly characterized. Gain or loss of function of Src-family kinases (SFKs) disassembles AChR clusters at adult NMJs in vivo, whereas AChR aggregates disperse rapidly upon withdrawal of agrin from cultured src -/-;fyn -/- myotubes. This suggests that a balance between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) such as those of the Src-family may be essential in stabilizing clusters of AChRs. Results We have analyzed the role of PTPs in maintenance of AChR aggregates, by adding and then withdrawing agrin from cultured myotubes in the presence of PTP or PTK inhibitors and quantitating remaining AChR clusters. In wild-type myotubes, blocking PTPs with pervanadate caused enhanced disassembly of AChR clusters after agrin withdrawal. When added at the time of agrin withdrawal, SFK inhibitors destabilized AChR aggregates but concomitant addition of pervanadate rescued cluster stability. Likewise in src -/-;fyn -/- myotubes, in which agrin-induced AChR clusters form normally but rapidly disintegrate after agrin withdrawal, pervanadate addition stabilized AChR clusters. The PTP SHP-2, known to be enriched at the NMJ, associated and colocalized with MuSK, and agrin increased this interaction. Specific SHP-2 knockdown by RNA interference reduced the stability of AChR clusters in wild-type myotubes. Similarly, knockdown of SHP-2 in adult mouse soleus muscle by electroporation of RNA interference constructs caused disassembly of pretzel-shaped AChR-rich areas in vivo. Finally, we found that src -/-;fyn -/- myotubes contained elevated levels of SHP-2 protein. Conclusion Our data are the first to show that the fine balance between PTPs and SFKs is a key aspect in stabilization of postsynaptic AChR clusters. One phosphatase that acts in this equilibrium is SHP-2. Thus, PTPs such as SHP-2 stabilize AChR clusters under normal circumstances, but when these PTPs are not balanced by SFKs, they render clusters unstable.
- Published
- 2007
30. Linker molecules between laminins and dystroglycan ameliorate laminin-alpha2-deficient muscular dystrophy at all disease stages
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Shuo Lin, Markus A. Rüegg, Sarina Meinen, Hanns Lochmüller, and Patrizia Barzaghi
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animal structures ,Recombinant Fusion Proteins ,Integrin ,Mice, Transgenic ,Perlecan ,Chick Embryo ,Basement Membrane ,Article ,Mice ,Laminin ,Dystroglycan ,medicine ,Animals ,Agrin ,Muscular dystrophy ,Dystroglycans ,Muscle, Skeletal ,Cells, Cultured ,Research Articles ,Binding Sites ,biology ,Cell Biology ,Genetic Therapy ,Muscular Dystrophy, Animal ,medicine.disease ,Molecular biology ,Cell biology ,Protein Structure, Tertiary ,Disease Models, Animal ,Treatment Outcome ,biology.protein ,Congenital muscular dystrophy ,Disease Progression ,Pikachurin ,Heparan Sulfate Proteoglycans - Abstract
Mutations in laminin-alpha2 cause a severe congenital muscular dystrophy, called MDC1A. The two main receptors that interact with laminin-alpha2 are dystroglycan and alpha7beta1 integrin. We have previously shown in mouse models for MDC1A that muscle-specific overexpression of a miniaturized form of agrin (mini-agrin), which binds to dystroglycan but not to alpha7beta1 integrin, substantially ameliorates the disease (Moll, J., P. Barzaghi, S. Lin, G. Bezakova, H. Lochmuller, E. Engvall, U. Muller, and M.A. Ruegg. 2001. Nature. 413:302-307; Bentzinger, C.F., P. Barzaghi, S. Lin, and M.A. Ruegg. 2005. Matrix Biol. 24:326-332.). Now we show that late-onset expression of mini-agrin still prolongs life span and improves overall health, although not to the same extent as early expression. Furthermore, a chimeric protein containing the dystroglycan-binding domain of perlecan has the same activities as mini-agrin in ameliorating the disease. Finally, expression of full-length agrin also slows down the disease. These experiments are conceptual proof that linking the basement membrane to dystroglycan by specifically designed molecules or by endogenous ligands, could be a means to counteract MDC1A at a progressed stage of the disease, and thus opens new possibilities for the development of treatment options for this muscular dystrophy.
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- 2007
31. Synapse loss in cortex of agrin-deficient mice after genetic rescue of perinatal death
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Riad Seddik, Iwona Ksiazek, Pico Caroni, Silvia Arber, Mathias Jucker, Joshua R. Sanes, Constanze Burkhardt, Gabriela Bezakova, Marcin Maj, Bernhard Bettler, Shuo Lin, and Markus A. Rüegg
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Male ,animal structures ,MAP Kinase Signaling System ,Synaptogenesis ,Mice, Transgenic ,Biology ,Neuromuscular junction ,Synapse ,Mice ,Excitatory synapse ,Postsynaptic potential ,medicine ,Animals ,Agrin ,Cerebral Cortex ,General Neuroscience ,Age Factors ,Articles ,Survival Rate ,medicine.anatomical_structure ,Animals, Newborn ,nervous system ,Silent synapse ,Synapses ,Excitatory postsynaptic potential ,Female ,Neuroscience ,Chickens - Abstract
Agrin-deficient mice die at birth because of aberrant development of the neuromuscular junctions. Here, we examined the role of agrin at brain synapses. We show that agrin is associated with excitatory but not inhibitory synapses in the cerebral cortex. Most importantly, we examined the brains of agrin-deficient mice whose perinatal death was prevented by the selective expression of agrin in motor neurons. We find that the number of presynaptic and postsynaptic specializations is strongly reduced in the cortex of 5- to 7-week-old mice. Consistent with a reduction in the number of synapses, the frequency of miniature postsynaptic currents was greatly decreased. In accordance with the synaptic localization of agrin to excitatory synapses, changes in the frequency were only detected for excitatory but not inhibitory synapses. Moreover, we find that the muscle-specific receptor tyrosine kinase MuSK, which is known to be an essential component of agrin-induced signaling at the neuromuscular junction, is also localized to a subset of excitatory synapses. Finally, some components of the mitogen-activated protein (MAP) kinase pathway, which has been shown to be activated by agrin in cultured neurons, are deregulated in agrin-deficient mice. In summary, our results provide strong evidence that agrin plays an important role in the formation and/or the maintenance of excitatory synapses in the brain, and we provide evidence that this function involves MAP kinase signaling.
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- 2007
32. Agrin is highly expressed by chondrocytes and is required for normal growth
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Rolf E. Brenner, Iwona Ksiazek, Markus A. Rüegg, and Heinz-Juergen Hausser
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Male ,medicine.medical_specialty ,Histology ,animal structures ,Transgene ,Gene Expression ,Apoptosis ,Mice, Transgenic ,Cartilage metabolism ,Biology ,Chondrocyte ,Neuromuscular junction ,Mice ,Chondrocytes ,Internal medicine ,medicine ,Animals ,Aggrecans ,Agrin ,Growth Plate ,Molecular Biology ,Collagen Type II ,Aggrecan ,Growth Disorders ,Cell Proliferation ,Mice, Knockout ,Cell growth ,Reverse Transcriptase Polymerase Chain Reaction ,Cartilage ,Receptor Protein-Tyrosine Kinases ,Cell Biology ,Immunohistochemistry ,Medical Laboratory Technology ,medicine.anatomical_structure ,Endocrinology ,nervous system ,Female ,Chickens - Abstract
Agrin is a heparan sulfate proteoglycan that is best known for its crucial involvement in the organization and maintenance of postsynaptic structures at the neuromuscular junction. Consistent with this role, mice deficient of agrin die at birth due to respiratory failure. Here we examined the early postnatal development of agrin-deficient mice in which perinatal death was prevented by transgenic expression of neural agrin in motor neurons. Such transgenic, agrin-deficient mice were born at Mendelian ratio but exhibited severe postnatal growth retardation. Growth plate morpholgy was markedly altered in these mice, with changes being most prominent in the hypertrophic zone. Compression of this zone was not caused by reduced viability of hypertrophic chondrocytes, as no differences in the apoptosis rates could be observed. Furthermore, deposition of the major cartilage matrix components collagen type II and aggrecan was slightly reduced in these mice. Consistent with a role for agrin in skeletal development, we show for the first time that agrin is highly expressed by chondrocytes and localizes to the growth plate in wild-type mice. Our data show that agrin is expressed in cartilage and that it plays a critical role in normal skeletal growth.
- Published
- 2007
33. Activation of muscle-specific receptor tyrosine kinase and binding to dystroglycan are regulated by alternative mRNA splicing of agrin
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Patrick Scotton, Jörg Stetefeld, Dorothee Bleckmann, Andrea Brancaccio, Francesca Sciandra, Thomas Meier, Michael Stebler, and Markus A. Rüegg
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Gene isoform ,animal structures ,Molecular Sequence Data ,Biochemistry ,Neuromuscular junction ,Mice ,Laminin ,medicine ,Dystroglycan ,Animals ,Humans ,Agrin ,Amino Acid Sequence ,RNA, Messenger ,Dystroglycans ,Molecular Biology ,chemistry.chemical_classification ,Binding Sites ,biology ,Sequence Homology, Amino Acid ,Kinase ,Muscles ,Receptor Protein-Tyrosine Kinases ,Cell Biology ,Amino acid ,Alternative Splicing ,medicine.anatomical_structure ,nervous system ,chemistry ,biology.protein ,Phosphorylation ,Chickens - Abstract
Agrin induces the aggregation of postsynaptic proteins at the neuromuscular junction (NMJ). This activity requires the receptor-tyrosine kinase MuSK. Agrin isoforms differ in short amino acid stretches at two sites, called A and B, that are localized in the two most C-terminal laminin G (LG) domains. Importantly, agrin isoforms greatly differ in their activities of inducing MuSK phosphorylation and of binding to alpha-dystroglycan. By using site-directed mutagenesis, we characterized the amino acids important for these activities of agrin. We find that the conserved tripeptide asparagineglutamate- isoleucine in the eight-amino acid long insert at the B-site is necessary and sufficient for full MuSK phosphorylation activity. However, even if all eight amino acids were replaced by alanines, this agrin mutant still has significantly higher MuSK phosphorylation activity than the splice version lacking any insert. We also show that binding to alpha-dystroglycan requires at least two LG domains and that amino acid inserts at the A and the B splice sites negatively affect binding.
- Published
- 2006
34. Clustering transmembrane-agrin induces filopodia-like processes on axons and dendrites
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Godela Bittcher, Maik Annies, Jürgen Löschinger, Elmar Porten, Stefan Wöll, Rene Ramseger, Stephan Kröger, Christian Abraham, and Markus A. Rüegg
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animal structures ,Dendritic spine ,Time Factors ,Neurite ,Cytochalasin B ,Growth Cones ,Video microscopy ,Chick Embryo ,Biology ,Nervous System ,Antibodies ,Cellular and Molecular Neuroscience ,Mice ,medicine ,Neurites ,Animals ,Agrin ,Pseudopodia ,Growth cone ,Cytoskeleton ,Molecular Biology ,Cells, Cultured ,Microscopy, Video ,Dose-Response Relationship, Drug ,Cell Membrane ,Cell Differentiation ,Cell Biology ,Dendrites ,Cell biology ,medicine.anatomical_structure ,nervous system ,Animals, Newborn ,Neuron ,Filopodia - Abstract
The transmembrane form of agrin (TM-agrin) is primarily expressed in the CNS, particularly on neurites. To analyze its function, we clustered TM-agrin on neurons using anti-agrin antibodies. On axons from the chick CNS and PNS as well as on axons and dendrites from mouse hippocampal neurons anti-agrin antibodies induced the dose- and time-dependent formation of numerous filopodia-like processes. The processes appeared within minutes after antibody addition and contained a complex cytoskeleton. Formation of processes required calcium, could be inhibited by cytochalasine D, but was not influenced by staurosporine, heparin or pervanadate. Time-lapse video microscopy revealed that the processes were dynamic and extended laterally along the entire length of the neuron. The lateral processes had growth cones at their tips that initially adhered to the substrate, but subsequently collapsed and were retracted. These data provide the first evidence for a specific role of TM-agrin in shaping the cytoskeleton of neurites in the developing nervous system.
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- 2006
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35. Overexpression of mini-agrin in skeletal muscle increases muscle integrity and regenerative capacity in laminin-alpha2-deficient mice
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Shuo Lin, Patrizia Barzaghi, C. Florian Bentzinger, and Markus A. Rüegg
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animal structures ,Mice, Transgenic ,Biology ,Motor Activity ,Biochemistry ,Basement Membrane ,Mice ,Sarcolemma ,Genetics ,medicine ,Animals ,Regeneration ,Agrin ,Muscular dystrophy ,Receptor ,Dystroglycans ,Muscle, Skeletal ,Molecular Biology ,Mice, Knockout ,Reverse Transcriptase Polymerase Chain Reaction ,Skeletal muscle ,Anatomy ,Muscular Dystrophy, Animal ,medicine.disease ,Immunohistochemistry ,Cell biology ,medicine.anatomical_structure ,nervous system ,Gene Expression Regulation ,Congenital muscular dystrophy ,Basal lamina ,Laminin ,ITGA7 ,Biotechnology - Abstract
Mutations in the gene encoding the alpha2 subunit of laminins cause the severe "merosin-deficient congenital muscular dystrophy" (MDC1A). We have recently shown that overexpression of a miniaturized form of the molecule agrin (mini-agrin) counteracts the disease in dy(W)/dy(W) mice, a model for MDC1A. However, these mice express some residual truncated laminin-alpha2, suggesting that the observed amelioration might be due to mini-agrin's presenting the residual laminin-alpha2 to its receptors. Here we show that the mini-agrin counteracts the disease in dy(3K)/dy(3K) mice, which are null for laminin-alpha2. As in dy(W)/dy(W) mice, mini-agrin improves both the function and structure of muscle. We show that muscle regeneration after injury is severely impaired in dy(3K)/dy(3K) mice but is restored in the mini-agrin-expressing littermates. In summary, our results 1) show that the direct linkage of muscle basal lamina with the sarcolemma is the basis of mini-agrin-mediated amelioration and 2) provide unprecedented evidence that this linkage is important for proper regeneration of muscle fibers after injury. Our findings thus suggest that treatment with mini-agrin might be beneficial over the entire spectrum of the MDC1A disease, whose severity inversely correlates with expression levels and the size of the truncation in laminin-alpha2.
- Published
- 2005
36. Src-family kinases stabilize the neuromuscular synapse in vivo via protein interactions, phosphorylation, and cytoskeletal linkage of acetylcholine receptors
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Xian Chu Kong, Gayathri Sadasivam, Susanne Erb-Vögtli, Markus A. Rüegg, Christian Fuhrer, Shuo Lin, Raffaella Willmann, University of Zurich, and Fuhrer, Christian
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Time Factors ,Utrophin ,Muscle Fibers, Skeletal ,Muscle Proteins ,Proto-Oncogene Proteins c-fyn ,Mice ,chemistry.chemical_compound ,Neurofilament Proteins ,Postsynaptic potential ,Receptors, Cholinergic ,Phosphorylation ,Dystroglycans ,Cells, Cultured ,Cytoskeleton ,Microscopy, Confocal ,Agrin ,General Neuroscience ,Dysbindin ,2800 General Neuroscience ,musculoskeletal system ,Immunohistochemistry ,Cell biology ,Electroporation ,src-Family Kinases ,medicine.anatomical_structure ,RNA Interference ,tissues ,Cellular/Molecular ,Proto-oncogene tyrosine-protein kinase Src ,animal structures ,Green Fluorescent Proteins ,Immunoblotting ,Neuromuscular Junction ,Synaptophysin ,610 Medicine & health ,In Vitro Techniques ,Biology ,Models, Biological ,Neuromuscular junction ,Imaging, Three-Dimensional ,FYN ,medicine ,Animals ,Acetylcholine receptor ,10242 Brain Research Institute ,Tyrosine phosphorylation ,Bungarotoxins ,Gene Expression Regulation ,chemistry ,Mutagenesis ,Dystrophin-Associated Proteins ,570 Life sciences ,biology ,Carrier Proteins ,Neuroscience - Abstract
Postnatal stabilization and maturation of the postsynaptic membrane are important for development and function of the neuromuscular junction (NMJ), but the underlying mechanisms remain poorly characterized. We examined the role of Src-family kinases (SFKs)in vivo. Electroporation of kinase-inactive Src constructs into soleus muscles of adult mice caused NMJ disassembly: acetylcholine receptor (AChR)-rich areas became fragmented; the topology of nerve terminal, AChRs, and synaptic nuclei was disturbed; and occasionally nerves started to sprout. Electroporation of kinase-overactive Src produced similar but milder effects. We studied the mechanism of SFK action using culturedsrc-/-;fyn-/-myotubes, focusing on clustering of postsynaptic proteins, their interaction with AChRs, and AChR phosphorylation. Rapsyn and the utrophin-glycoprotein complex were recruited normally into AChR-containing clusters by agrin insrc-/-;fyn-/-myotubes. But after agrin withdrawal, clusters of these proteins disappeared rapidly in parallel with AChRs, revealing that SFKs are of general importance in postsynaptic stability. At the same time, AChR interaction with rapsyn and dystrobrevin and AChR phosphorylation decreased after agrin withdrawal from mutant myotubes. Unexpectedly, levels of rapsyn protein were increased insrc-/-;fyn-/-myotubes, whereas rapsyn-cytoskeleton interactions were unaffected. The overall cytoskeletal link of AChRs was weak but still strengthened by agrin in mutant cells, consistent with the normal formation but decreased stability of AChR clusters. These data show that correctly balanced activity of SFKs is critical in maintaining adult NMJsin vivo. SFKs hold the postsynaptic apparatus together through stabilization of AChR-rapsyn interaction and AChR phosphorylation. In addition, SFKs control rapsyn levels and AChR-cytoskeletal linkage.
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- 2005
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37. Tyrosine phosphatase regulation of MuSK-dependent acetylcholine receptor clustering
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Markus A. Rüegg, H. Benjamin Peng, Xiaotao T. Zhao, and Raghavan Madhavan
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animal structures ,Xenopus ,Muscle Fibers, Skeletal ,Neuromuscular Junction ,Down-Regulation ,Protein Tyrosine Phosphatase, Non-Receptor Type 11 ,Protein tyrosine phosphatase ,Synaptic Transmission ,Neuromuscular junction ,Receptor tyrosine kinase ,Cellular and Molecular Neuroscience ,Mice ,medicine ,Animals ,Receptors, Cholinergic ,Agrin ,Tyrosine ,Enzyme Inhibitors ,Muscle, Skeletal ,Molecular Biology ,Cells, Cultured ,Acetylcholine receptor ,biology ,Myogenesis ,Receptor Aggregation ,Intracellular Signaling Peptides and Proteins ,Receptor Protein-Tyrosine Kinases ,Cell Biology ,Cell biology ,medicine.anatomical_structure ,nervous system ,Biochemistry ,biology.protein ,Protein Tyrosine Phosphatases ,Vanadates ,Tyrosine kinase - Abstract
During vertebrate neuromuscular junction (NMJ) development, nerve-secreted agrin induces acetylcholine receptor (AChR) clustering in muscle by activating the muscle-specific tyrosine kinase MuSK. Recently, it has been recognized that MuSK activation-dependent AChR clustering occurs in embryonic muscle even in the absence of agrin, but how this process is regulated is poorly understood. We report that inhibition of tyrosine phosphatases in cultured C2 mouse myotubes using pervanadate enhanced MuSK auto-activation and agrin-independent AChR clustering. Moreover, phosphatase inhibition also enlarged the AChR clusters induced by agrin in these cells. Conversely, in situ activation of MuSK in cultured Xenopus embryonic muscle cells, either focally by anti-MuSK antibody-coated beads or globally by agrin, stimulated downstream tyrosine phosphatases, which could be blocked by pervanadate treatment. Immunoscreening identified Shp2 as a major tyrosine phosphatase in C2 myotubes and down-regulation of its expression by RNA interference alleviated tyrosine phosphatase suppression of MuSK activation. Significantly, depletion of Shp2 increased both agrin-independent and agrin-dependent AChR clustering in myotubes. Our results suggest that muscle tyrosine phosphatases tightly regulate MuSK activation and signaling and support a novel role of Shp2 in MuSK-dependent AChR clustering.
- Published
- 2004
38. Expression of mouse agrin in normal, denervated and dystrophic muscle
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Patrizia Barzaghi, Alexander Eusebio, Filippo Oliveri, and Markus A. Rüegg
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Gene isoform ,animal structures ,Blotting, Western ,Gene Expression ,Neuromuscular junction ,Muscular Dystrophies ,Dystrophin ,Mice ,Laminin ,Postsynaptic potential ,medicine ,Animals ,Agrin ,Muscle, Skeletal ,Genetics (clinical) ,biology ,medicine.disease ,Molecular biology ,Denervation ,Precipitin Tests ,Mice, Mutant Strains ,medicine.anatomical_structure ,nervous system ,Neurology ,Pediatrics, Perinatology and Child Health ,biology.protein ,Congenital muscular dystrophy ,Mice, Inbred mdx ,Neurology (clinical) ,ITGA7 ,Integrin alpha Chains ,Heparan Sulfate Proteoglycans - Abstract
Agrin is a heparan sulfate proteoglycan that is required for the development of postsynaptic specializations at the neuromuscular junction. An alternatively spliced isoform of agrin that lacks this activity is found in basement membranes of several tissues including embryonic muscle. Overexpression of a miniaturized form of this agrin isoform ameliorates the severe muscle dystrophy of laminin alpha2-deficient mice, a mouse model for merosin-deficient congenital muscle dystrophy. Several lines of evidence indicate that this amelioration is based on the high-affinity binding of the mini-agrin to the laminins and to alpha-dystroglycan. Here, we used antibodies raised against mouse agrin to evaluate protein expression in adult muscle of normal and dystrophic mice. We find that expression of agrin in non-synaptic region varies greatly between different muscles in wild-type mice and that its levels are altered in dystrophic muscle.
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- 2003
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39. A neuronal inhibitory domain in the N-terminal half of agrin
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John L, Bixby, Kristine, Baerwald-De la Torre, Cong, Wang, Fritz G, Rathjen, and Markus A, Rüegg
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Motor Neurons ,Integrins ,Binding Sites ,Membrane Glycoproteins ,Cell Adhesion Molecules, Neuronal ,Immunoglobulins ,Protein Structure, Tertiary ,Cytoskeletal Proteins ,Contactins ,Cell Adhesion ,Contactin 2 ,Neurites ,Animals ,Agrin ,Nerve Growth Factors ,Dystroglycans ,Cell Adhesion Molecules ,Neural Cell Adhesion Molecules ,Cell Division ,Cells, Cultured - Abstract
Agrin is required for appropriate pre- and postsynaptic differentiation of neuromuscular junctions. While agrin's ability to orchestrate postsynaptic differentiation is well documented, more recent experiments have suggested that agrin is also a "stop signal" for the presynaptic neuron, and that agrin has actions on neurons in the CNS. To elucidate the neuronal activities of agrin and to define the receptor(s) responsible for these functions, we have examined adhesions of neurons and their neurite-outgrowth responses to purified agrin in vitro. We find that both full-length agrin and the C-terminal 95 kDa of agrin (agrin c95), which is sufficient to induce postsynaptic differentiation, are adhesive for chick ciliary ganglion (CG) and forebrain neurons. Consistent with previous findings, our results show that N-CAM binds to full-length agrin, and suggest that alpha-dystroglycan is a neuronal receptor for agrin c95. In neurite outgrowth assays, full-length agrin inhibited both laminin- and N-cadherin-induced neurite growth from CG neurons. The N-terminal 150 kDa fragment of agrin, but not agrin c95, inhibited neurite outgrowth, indicating that domains in the N-terminal portion of agrin are sufficient for this function. Adhesion assays using protein-coated beads and agrin-expressing cells revealed differential interactions of agrin with members of the immunoglobulin superfamily of cell adhesion molecules. However, none of these, including N-CAM, appeared to be critical for neuronal adhesion. In summary, our results suggest that the N-terminal half of agrin is involved in agrin's ability to inhibit neurite outgrowth. Our results further suggest that neither alpha-dystroglycan nor N-CAM, two known binding proteins for agrin, mediate this effect.
- Published
- 2002
40. An agrin minigene rescues dystrophic symptoms in a mouse model for congenital muscular dystrophy
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Shuo Lin, Hanns Lochmüller, Eva Engvall, Markus A. Rüegg, Patrizia Barzaghi, Gabriela Bezakova, Ulrich Müller, and Joachim Moll
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medicine.medical_specialty ,Neuromuscular junction ,Basement Membrane ,Mice ,Laminin ,Internal medicine ,medicine ,Animals ,Agrin ,Muscular dystrophy ,Dystroglycans ,Muscle, Skeletal ,Basement membrane ,Multidisciplinary ,Membrane Glycoproteins ,biology ,Muscular Dystrophy, Animal ,medicine.disease ,Cell biology ,Cytoskeletal Proteins ,Disease Models, Animal ,medicine.anatomical_structure ,Endocrinology ,biology.protein ,Congenital muscular dystrophy ,ITGA7 ,Chickens ,Minigene ,Protein Binding - Abstract
Congenital muscular dystrophy is a heterogeneous and severe, progressive muscle-wasting disease that frequently leads to death in early childhood. Most cases of congenital muscular dystrophy are caused by mutations in LAMA2, the gene encoding the alpha2 chain of the main laminin isoforms expressed by muscle fibres. Muscle fibre deterioration in this disease is thought to be caused by the failure to form the primary laminin scaffold, which is necessary for basement membrane structure, and the missing interaction between muscle basement membrane and the dystrophin-glycoprotein complex (DGC) or the integrins. With the aim to restore muscle function in a mouse model for this disease, we have designed a minigene of agrin, a protein known for its role in the formation of the neuromuscular junction. Here we show that this mini-agrin-which binds to basement membrane and to alpha-dystroglycan, a member of the DGC-amends muscle pathology by a mechanism that includes agrin-mediated stabilization of alpha-dystroglycan and the laminin alpha5 chain. Our data provides in vivo evidence that a non-homologous protein in combination with rational protein design can be used to devise therapeutic tools that may restore muscle function in human muscular dystrophies.
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- 2001
41. An alternative amino-terminus expressed in the central nervous system converts agrin to a type II transmembrane protein
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Godela Bittcher, Maik Annies, Stephan Kröger, Beat Schumacher, Frank Neumann, and Markus A. Rüegg
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Signal peptide ,Central Nervous System ,animal structures ,Glycosylation ,Neuromuscular Junction ,Chick Embryo ,Protein Sorting Signals ,Transfection ,Neuromuscular junction ,Cell Line ,Cellular and Molecular Neuroscience ,Mice ,Species Specificity ,Complementary DNA ,medicine ,Animals ,Humans ,Receptors, Cholinergic ,Agrin ,Molecular Biology ,Secretory pathway ,Conserved Sequence ,biology ,Sequence Homology, Amino Acid ,Cell Membrane ,Receptor Aggregation ,Membrane Proteins ,Cell Biology ,Transmembrane protein ,Rats ,medicine.anatomical_structure ,nervous system ,Proteoglycan ,Biochemistry ,biology.protein ,Basal lamina ,Protein Processing, Post-Translational - Abstract
Agrin is a basal lamina-associated heparansulfate proteoglycan that is a key molecule in the formation of the vertebrate neuromuscular junction. The carboxy-terminal part of agrin is involved in its synaptogenic activity. The amino-terminal end of chick agrin consists of a signal sequence, required for the targeting of the protein to the secretory pathway, and the amino-terminal agrin (NtA) domain that binds to basal lamina-associated laminins. The cDNA encoding rat agrin lacks this NtA domain and instead codes for a shorter amino-terminal end. While the NtA domain is conserved in several species, including human, sequences homologous to the amino-terminus of rat agrin have not been described. In this work, we have characterized these amino-terminal sequences in mouse and chick. We show that they all serve as a noncleaved signal anchor that immobilizes the protein in a N(cyto)/C(exo) orientation in the plasma membrane. Like the secreted form, this transmembrane form of agrin is highly glycosylated indicative of a heparansulfate proteoglycan. The structure of the 5' end of the mouse agrin gene suggests that a distinct promoter drives expression of the transmembrane form. Agrin transcripts encoding this form are enriched in the embryonic brain, particularly in neurons. To our knowledge, this is the first example of a molecule that is synthesized both as a basal lamina and a plasma membrane protein.
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- 2001
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42. The Ets Transcription Factor GABP Is Required for Postsynaptic Differentiation In Vivo
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Alexandre Briguet and Markus A. Rüegg
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animal structures ,Utrophin ,Muscle Fibers, Skeletal ,Neuromuscular Junction ,Synaptic Membranes ,Biology ,Neuromuscular junction ,Neurotransmitter receptor ,Postsynaptic potential ,medicine ,Animals ,Receptors, Cholinergic ,Agrin ,ARTICLE ,Promoter Regions, Genetic ,Transcription factor ,Acetylcholine receptor ,General Neuroscience ,Gene Expression Regulation, Developmental ,Membrane Proteins ,Cell Differentiation ,GA-Binding Protein Transcription Factor ,Cell biology ,Rats ,DNA-Binding Proteins ,Cytoskeletal Proteins ,medicine.anatomical_structure ,COS Cells ,Mutation ,Acetylcholinesterase ,Neuroscience ,Postsynaptic density ,Transcription Factors - Abstract
At chemical synapses, neurotransmitter receptors are concentrated in the postsynaptic membrane. During the development of the neuromuscular junction, motor neurons induce aggregation of acetylcholine receptors (AChRs) underneath the nerve terminal by the redistribution of existing AChRs and preferential transcription of the AChR subunit genes in subsynaptic myonuclei. Neural agrin, when expressed in nonsynaptic regions of muscle fibers in vivo, activates both mechanisms resulting in the assembly of a fully functional postsynaptic apparatus. Several lines of evidence indicate that synaptic transcription of AChR genes is primarily dependent on a promoter element called N-box. The Ets-related transcription factor growth-associated binding protein (GABP) binds to this motif and has thus been suggested to regulate synaptic gene expression. Here, we assessed the role of GABP in synaptic gene expression and in the formation of postsynaptic specializations in vivo by perturbing its function during postsynaptic differentiation induced by neural agrin. We find that neural agrin-mediated activation of the AChR epsilon subunit promoter is abolished by the inhibition of GABP function. Importantly, the number of AChR aggregates formed in response to neural agrin was strongly reduced. Moreover, aggregates of acetylcholine esterase and utrophin, two additional components of the postsynaptic apparatus, were also reduced. Together, these results are the first direct in vivo evidence that GABP regulates synapse-specific gene expression at the neuromuscular junction and that GABP is required for the formation of a functional postsynaptic apparatus.
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- 2000
43. Composition, synthesis, and assembly of the embryonic chick retinal basal lamina
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Andreas Osanger, W. Schneider, Gregory J. Cole, Markus A. Rüegg, Sucai Dong, Willi Halfter, and Barbara Schurer
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retina ,Time Factors ,genetic structures ,extracellular matrix ,Blotting, Western ,Nervous System ,basal lamina ,Basement Membrane ,03 medical and health sciences ,chemistry.chemical_compound ,Basal (phylogenetics) ,Laminin ,Culture Techniques ,Anchoring fibrils ,medicine ,Animals ,Regeneration ,Agrin ,Collagenases ,chick embryo ,Molecular Biology ,In Situ Hybridization ,030304 developmental biology ,vitreous body ,0303 health sciences ,Retina ,Membrane Glycoproteins ,biology ,030302 biochemistry & molecular biology ,Retinal ,Tenascin ,Anatomy ,Cell Biology ,Lamina lucida ,eye diseases ,Cell biology ,medicine.anatomical_structure ,chemistry ,Microscopy, Fluorescence ,biology.protein ,Lamina densa ,Basal lamina ,Proteoglycans ,Collagen ,Heparitin Sulfate ,sense organs ,Heparan Sulfate Proteoglycans ,Developmental Biology - Abstract
To study the biology of basal laminae in the developing nervous system the protein composition of the embryonic retinal basal lamina was investigated, the site of synthesis of its proteins in the eye was determined, and basal lamina assembly was studied in vivo in two assay systems. Laminin, nidogen, agrin, collagen IV, and XVIII are major constituents of the retinal basal lamina. However, only agrin is synthesized by the retina, whereas the other matrix constituents originate from cells of the ciliary body, the lens, or the optic disc. The synthesis from extraretinal tissues infers that the retinal basal lamina proteins must be shed from their tissues of origin into the vitreous body and from there bind to receptor proteins provided by the retinal neuroepithelium. The fact that all proteins typical for the retinal basal lamina are abundant in the vitreous body and a new basal lamina is only formed when the vitreous body was directly adjacent to the retina is consistent with the contention of the vitreous body having a function in retinal basal lamina formation. Basal lamina assembly was also studied after disrupting the retinal basal lamina by intraocular injection of collagenase. The basal lamina regenerated after chasing the collagenase with Matrigel, which served as a collagenase inhibitor. The basal lamina was reconstituted within 6 h. However, the regenerated basal lamina was located deeper in the retina than normal by reconstituting along the retracted neuroepithelial endfeet demonstrating that these endfeet are the preferred site of basal lamina assembly.
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- 2000
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44. Interaction of agrin with laminin requires a coiled-coil conformation of the agrin-binding site within the laminin gamma1 chain
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Markus A. Rüegg, Jürgen Engel, Alain J. Denzer, Peter D. Yurchenco, Richard A. Kammerer, Therese Schulthess, Ruth Landwehr, Ariel Lustig, and Beat Schumacher
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animal structures ,DNA, Complementary ,Protein Conformation ,Protein subunit ,Molecular Sequence Data ,Plasma protein binding ,Transfection ,General Biochemistry, Genetics and Molecular Biology ,Protein–protein interaction ,Protein structure ,Laminin ,Escherichia coli ,Animals ,Agrin ,Amino Acid Sequence ,Binding site ,Molecular Biology ,Coiled coil ,Binding Sites ,General Immunology and Microbiology ,biology ,Sequence Homology, Amino Acid ,General Neuroscience ,Circular Dichroism ,Temperature ,Recombinant Proteins ,Protein Structure, Tertiary ,Biochemistry ,COS Cells ,biology.protein ,Biophysics ,Ultracentrifugation ,Gene Deletion ,Research Article ,Protein Binding - Abstract
Coiled-coil domains are found in a wide variety of proteins, where they typically specify subunit oligomerization. Recently, we have demonstrated that agrin, a multidomain heparan sulfate proteoglycan with a crucial role in the development of the nerve-muscle synapse, binds to the three-stranded coiled-coil domain of laminin-1. The interaction with laminin mediates the integration of agrin into basement membranes. Here we characterize the binding site within the laminin-1 coiled coil in detail. Binding assays with individual laminin-1 full-length chains and fragments revealed that agrin specifically interacts with the gamma1 subunit of laminin-1, whereas no binding to alpha1 and beta1 chains was detected. By using recombinant gamma1 chain fragments, we mapped the binding site to a sequence of 20 residues. Furthermore, we demonstrate that a coiled-coil conformation of this binding site is required for its interaction with agrin. The finding that recombinant gamma1 fragments bound at least 10-fold less than native laminin-1 indicates that the structure of the three-stranded coiled-coil domain of laminin is required for high-affinity agrin binding. Interestingly, no binding to a chimeric gamma2 fragment was observed, indicating that the interaction of agrin with laminin is isoform specific.
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- 1999
45. Characterisation of alpha-dystrobrevin in muscle
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Nellie Y. Loh, Derek J. Blake, Kay E. Davies, Markus A. Rüegg, and Ralph Nawrotzki
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Utrophin ,Molecular Sequence Data ,Muscle Proteins ,Biology ,Neuromuscular junction ,Cell Line ,Mice ,Fetus ,Dystrobrevin ,medicine ,Myocyte ,Animals ,Humans ,Protein Isoforms ,Agrin ,Amino Acid Sequence ,Muscle, Skeletal ,Phosphotyrosine ,Acetylcholine receptor ,Base Sequence ,Neuropeptides ,Skeletal muscle ,Brain ,Membrane Proteins ,Cell Biology ,musculoskeletal system ,Molecular biology ,Cell biology ,Cytoskeletal Proteins ,Disease Models, Animal ,medicine.anatomical_structure ,Dystrophin-Associated Proteins ,Synapses ,biology.protein ,Mice, Inbred mdx ,Vanadates ,Dystrophin - Abstract
Dystrophin-related and associated proteins are important for the formation and maintenance of the mammalian neuromuscular junction. Initial studies in the electric organ of Torpedo californica showed that the dystrophin-related protein dystrobrevin (87K) co-purifies with the acetylcholine receptors and other postsynaptic proteins. Dystrobrevin is also a major phosphotyrosine-containing protein in the postsynaptic membrane. Since inhibitors of tyrosine protein phosphorylation block acetylcholine receptor clustering in cultured muscle cells, we examined the role of alpha-dystrobrevin during synapse formation and in response to agrin. Using specific antibodies, we show that C2 myoblasts and early myotubes only produce alpha-dystrobrevin-1, the mammalian orthologue of Torpedo dystrobrevin, whereas mature skeletal muscle expresses three distinct alpha-dystrobrevin isoforms. In myotubes, alpha-dystrobrevin-1 is found on the cell surface and also in acetylcholine receptor-rich domains. Following agrin stimulation, alpha-dystrobrevin-1 becomes re-localised beneath the cell surface into macroclusters that contain acetylcholine receptors and another dystrophin-related protein, utrophin. This redistribution is not associated with tyrosine phosphorylation of alpha-dystrobrevin-1 by agrin. Furthermore, we show that alpha-dystrobrevin-1 is associated with both utrophin in C2 cells and dystrophin in mature skeletal muscle. Thus alpha-dystrobrevin-1 is a component of two protein complexes in muscle, one with utrophin at the neuromuscular junction and the other with dystrophin at the sarcolemma. These results indicate that alpha-dystrobrevin-1 is not involved in the phosphorylation-dependent, early stages of receptor clustering, but rather in the stabilisation and maturation of clusters, possibly via an interaction with utrophin.
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- 1998
46. Muscle-specific agrin isoforms reduce phosphorylation of AChR gamma and delta subunits in cultured muscle cells
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Bruce G. Wallace, Markus A. Rüegg, and Thomas Meier
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animal structures ,Recombinant Fusion Proteins ,Muscle Proteins ,Nerve Tissue Proteins ,Biology ,Neuromuscular junction ,Cellular and Molecular Neuroscience ,chemistry.chemical_compound ,Structure-Activity Relationship ,medicine ,Myocyte ,Animals ,Receptors, Cholinergic ,Agrin ,Phosphorylation ,Dystroglycans ,Muscle, Skeletal ,Molecular Biology ,Cells, Cultured ,Acetylcholine receptor ,Membrane Glycoproteins ,Myogenesis ,Tyrosine phosphorylation ,Cell Biology ,musculoskeletal system ,Peptide Fragments ,Cell biology ,Cytoskeletal Proteins ,medicine.anatomical_structure ,nervous system ,Biochemistry ,chemistry ,Laminin ,tissues ,C2C12 ,Chickens ,Protein Processing, Post-Translational - Abstract
The accumulation of nicotinic acetylcholine receptors (AChRs) at neuromuscular synapses is triggered by agrin, a protein that is synthesized by both nerve and muscle. Nerve-derived agrin, which contains an amino acid insert at a conserved splice site in the carboxy-terminal part of the protein, induces AChR aggregation and causes tyrosine phosphorylation of the AChR beta subunit. In contrast, agrin isoforms synthesized by muscle cells lack such an insert and have no effect on AChR distribution. In order to identify possible functional roles of muscle-derived agrin we have analyzed further the effect of various fragments of recombinant agrin on AChR phosphorylation. A carboxy-terminal fragment of muscle agrin, c95A0B0, reduced AChR gamma and delta subunit phosphorylation when added to C2C12 myotubes in culture. Although c95A0B0 had no effect on AChR beta subunit phosphorylation when added alone, it inhibited AChR beta subunit phosphorylation and AChR aggregation by the nerve-specific agrin isoform c95A4B8. We conclude that muscle-derived agrin can influence, both directly and indirectly, AChR phosphorylation. Such changes may play a role in the formation, maintenance, or function of the neuromuscular junction.
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- 1998
47. Evidence that agrin directly influences presynaptic differentiation at neuromuscular junctions in vitro
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Markus A. Rüegg, Jason A. Campagna, and John L. Bixby
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animal structures ,Neuromuscular Junction ,Nerve Tissue Proteins ,Chick Embryo ,Biology ,Transfection ,Synaptic vesicle ,Neuromuscular junction ,Synaptotagmin 1 ,Extracellular matrix ,Immunoglobulin Fab Fragments ,Postsynaptic potential ,medicine ,Animals ,Agrin ,Fluorescent Antibody Technique, Indirect ,Cells, Cultured ,Acetylcholine receptor ,Motor Neurons ,General Neuroscience ,Ciliary ganglion ,Cell Differentiation ,Immunohistochemistry ,Coculture Techniques ,Recombinant Proteins ,Cell biology ,medicine.anatomical_structure ,nervous system ,Immunoglobulin G ,COS Cells ,Synapses ,Neuroscience - Abstract
The synaptic protein agrin is required for aspects of both pre- and postsynaptic differentiation at neuromuscular junctions. Although a direct effect of agrin on postsynaptic differentiation, presumably through the MuSK receptor, is established, it is not clear whether agrin directly affects the presynaptic nerve. To provide evidence on this point, we used anti-agrin IgG to disrupt agrin function in chick ciliary ganglion (CG) neuron/myotube cocultures. In cocultures grown in the presence of 200 microg/ml anti-agrin IgG, clustering of acetylcholine receptors (AChRs), extracellular matrix proteins, and the synaptic vesicle protein synaptotagmin (syt) at nerve-muscle contacts was inhibited. Syt clustering was still inhibited in the presence of 100 microg/ml blocking antibody, while the postsynaptic clustering of AChRs, heparan sulphate proteoglycan, and s-laminin was retained. Additionally, in CG neurons cultured with COS cells expressing agrin A0B0, which lacks the ability to signal postsynaptic differentiation, syt clustering was induced and this clustering was also blocked by anti-agrin IgG. Our results demonstrate that agrin function is acutely required for pre- and postsynaptic differentiation in vitro, and strongly suggest that agrin is directly involved in the induction of presynaptic differentiation.
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- 1998
48. Agrin orchestrates synaptic differentiation at the vertebrate neuromuscular junction
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John L. Bixby and Markus A. Rüegg
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Neurons ,animal structures ,Agrin ,General Neuroscience ,Synaptogenesis ,Neuromuscular Junction ,Cell Differentiation ,Biology ,Neuromuscular junction ,Synapse ,medicine.anatomical_structure ,nervous system ,Postsynaptic potential ,Synapses ,medicine ,Dystroglycan ,biology.protein ,Neuregulin ,Animals ,Humans ,Neuroscience ,Transduction (physiology) - Abstract
The synapse is a key structure that is involved in perception, learning and memory. Understanding the sequence of steps that is involved in establishing synapses during development might also help to understand mechanisms that cause changes in synapses during learning and memory. For practical reasons, most of our current knowledge of synapse development is derived from studies of the vertebrate neuromuscular junction (NMJ). Several lines of evidence strongly suggest that motor axons release the molecule agrin to induce the formation of the postsynaptic apparatus in muscle fibers. Recent advances implicate proteins such as dystroglycan, MuSK, and rapsyn in the transduction of agrin signals. Recently, additional functions of agrin have been discovered, including the upregulation of gene transcription in myonuclei and the control of presynaptic differentiation. Agrin therefore appears to play a unique role in controlling synaptic differentiation on both sides of the NMJ.
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- 1998
49. Electron microscopic structure of agrin and mapping of its binding site in laminin-1
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Jürgen Engel, Richard A. Kammerer, Alain J. Denzer, Beat Schumacher, Therese Schulthess, Markus A. Rüegg, and Charlotte Fauser
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animal structures ,General Biochemistry, Genetics and Molecular Biology ,Neuromuscular junction ,Cell Line ,Extracellular matrix ,chemistry.chemical_compound ,Protein structure ,Laminin ,medicine ,Animals ,Humans ,Agrin ,Binding site ,Molecular Biology ,Binding Sites ,General Immunology and Microbiology ,biology ,General Neuroscience ,Heparan sulfate ,Peptide Fragments ,Recombinant Proteins ,Protein Structure, Tertiary ,Microscopy, Electron ,medicine.anatomical_structure ,Biochemistry ,chemistry ,nervous system ,COS Cells ,biology.protein ,Biophysics ,Basal lamina ,Chickens ,Research Article - Abstract
Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with basement membranes of several tissues. Particular splice variants of agrin are essential for the formation of synaptic structures at the neuromuscular junction. The binding of agrin to laminin appears to be required for its localization to synaptic basal lamina and other basement membranes. Here, electron microscopy was used to determine the structure of agrin and to localize its binding site in laminin-1. Agrin appears as an approximately 95 nm long particle that consists of a globular, N-terminal laminin-binding domain, a central rod predominantly formed by the follistatin-like domains and three globular, C-terminal laminin G-like domains. In a few cases, heparan sulfate glycosaminoglycan chains were seen emerging from the central portion of the core protein. Moreover, we show that agrin binds to the central region of the three-stranded, coiled-coil oligomerization domain in the long arm of laminin-1, which mediates subunit assembly of the native laminin molecule. In summary, our data show for the first time a protein-protein interaction of the extracellular matrix that involves a coiled-coil domain, and they assign a novel role to this domain of laminin-1. Based on this, we propose that agrin associates with basal lamina in a polarized way.
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- 1998
50. Agrin binds to the nerve-muscle basal lamina via laminin
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Markus A. Rüegg, Ralph Brandenberger, Alain J. Denzer, Matthias Gesemann, Matthias Chiquet, University of Zurich, and Ruegg, M A
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animal structures ,Molecular Sequence Data ,Neuromuscular Junction ,610 Medicine & health ,Chick Embryo ,Receptors, Nicotinic ,Neuromuscular junction ,Basement Membrane ,Retina ,Article ,Extracellular matrix ,1307 Cell Biology ,Mice ,Structure-Activity Relationship ,Laminin ,medicine ,Animals ,Humans ,Agrin ,Amino Acid Sequence ,Cells, Cultured ,Basement membrane ,Matrigel ,Binding Sites ,biology ,10242 Brain Research Institute ,Sequence Homology, Amino Acid ,Myogenesis ,Receptor Aggregation ,Cell Biology ,Molecular biology ,Peptide Fragments ,Cell biology ,Extracellular Matrix ,Drug Combinations ,medicine.anatomical_structure ,nervous system ,COS Cells ,biology.protein ,570 Life sciences ,Basal lamina ,Proteoglycans ,Collagen ,Sequence Alignment ,Protein Binding - Abstract
Agrin is a heparan sulfate proteoglycan that is required for the formation and maintenance of neuromuscular junctions. During development, agrin is secreted from motor neurons to trigger the local aggregation of acetylcholine receptors (AChRs) and other proteins in the muscle fiber, which together compose the postsynaptic apparatus. After release from the motor neuron, agrin binds to the developing muscle basal lamina and remains associated with the synaptic portion throughout adulthood. We have recently shown that full-length chick agrin binds to a basement membrane-like preparation called Matrigel™. The first 130 amino acids from the NH2 terminus are necessary for the binding, and they are the reason why, on cultured chick myotubes, AChR clusters induced by full-length agrin are small. In the current report we show that an NH2-terminal fragment of agrin containing these 130 amino acids is sufficient to bind to Matrigel™ and that the binding to this preparation is mediated by laminin-1. The fragment also binds to laminin-2 and -4, the predominant laminin isoforms of the muscle fiber basal lamina. On cultured myotubes, it colocalizes with laminin and is enriched in AChR aggregates. In addition, we show that the effect of full-length agrin on the size of AChR clusters is reversed in the presence of the NH2-terminal agrin fragment. These data strongly suggest that binding of agrin to laminin provides the basis of its localization to synaptic basal lamina and other basement membranes.
- Published
- 1997
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