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Markus A Ruegg - One of the best experts on this subject based on the ideXlab platform.

  • muscle wide secretion of a miniaturized form of neural Agrin rescues focal neuromuscular innervation in Agrin mutant mice
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Shuo Lin, Marcin Maj, Gabriela Bezakova, Josef P Magyar, Hans Rudolf Brenner, Markus A Ruegg
    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.

  • Expression of mouse Agrin in normal, denervated and dystrophic muscle
    Neuromuscular disorders : NMD, 2003
    Co-Authors: Alexander Eusebio, Patrizia Barzaghi, Filippo Oliveri, Markus A Ruegg
    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.

  • Agrin is a major heparan sulfate proteoglycan in the human glomerular basement membrane
    Journal of Histochemistry and Cytochemistry, 1998
    Co-Authors: Alexander J Groffen, Markus A Ruegg, Henry B P M Dijkman, T J A M Van De Velden, C A Buskens, J Van Den Born, Karel J M Assmann, Leo A H Monnens, J H Veerkamp, L P W J Van Den Heuvel
    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)

  • Alternative Splicing of Agrin Alters Its Binding to Heparin, Dystroglycan, and the Putative Agrin Receptor
    Neuron, 1996
    Co-Authors: Matthias Gesemann, Andrea Brancaccio, Valeria Cavalli, Alain J Denzer, Beat Schumacher, Markus A Ruegg
    Abstract:

    Agrin is a heparan sulfate proteoglycan that induces aggregation of acetylcholine receptors (AChRs) at the neuromuscular synapse. This aggregating activity is modulated by alternative splicing. Here, we compared binding of Agrin isoforms to heparin, alpha-dystroglycan, and cultured myotubes. We find that the alternatively spliced 4 amino acids insert (KSRK) is required for heparin binding. The binding affinity of Agrin isoforms to alpha-dystroglycan correlates neither with binding to heparin nor with their AChR-aggregating activities. Moreover, the minimal fragment sufficient to induce AChR aggregation does not bind to alpha-dystroglycan. Nevertheless, this fragment still binds to cultured muscle cells. Its binding is completed only by Agrin isoforms that are active in AChR aggregation, and therefore this binding site is likely to represent the receptor that initiates AChR clustering.

Julio Lopez - One of the best experts on this subject based on the ideXlab platform.

  • guess again and again and again measuring password strength by simulating password cracking algorithms
    IEEE Symposium on Security and Privacy, 2012
    Co-Authors: Patrick Gage Kelley, Michelle L Mazurek, Lujo Bauer, Nicolas Christin, Richard Shay, Timothy Vidas, Saranga Komanduri, Lorrie Faith Cranor, Julio Lopez
    Abstract:

    Text-based passwords remain the dominant authentication method in computer systems, despite significant advancement in attackers' capabilities to perform password cracking. In response to this threat, password composition policies have grown increasingly complex. However, there is insufficient research defining metrics to characterize password strength and using them to evaluate password-composition policies. In this paper, we analyze 12,000 passwords collected under seven composition policies via an online study. We develop an efficient distributed method for calculating how effectively several heuristic password-guessing algorithms guess passwords. Leveraging this method, we investigate (a) the resistance of passwords created under different conditions to guessing, (b) the performance of guessing algorithms under different training sets, (c) the relationship between passwords explicitly created under a given composition policy and other passwords that happen to meet the same requirements, and (d) the relationship between guess ability, as measured with password-cracking algorithms, and entropy estimates. Our findings advance understanding of both password-composition policies and metrics for quantifying password security.

  • guess again and again and again measuring password strength by simulating password cracking algorithms cmu cylab 11 008
    2011
    Co-Authors: Patrick Gage Kelley, Michelle L Mazurek, Lujo Bauer, Nicolas Christin, Richard Shay, Timothy Vidas, Saranga Komanduri, Lorrie Faith Cranor, Julio Lopez
    Abstract:

    Text-based passwords remain the dominant authentication method in computer systems, despite significant advancement in attackers’ capabilities to perform password cracking. In response to this threat, password composition policies have grown increasingly complex. However, there is insufficient research defining metrics to characterize password strength and evaluating password-composition policies using these metrics. In this paper, we describe an analysis of 12,000 passwords collected under seven composition policies via an online study. We develop an efficient distributed method for calculating how effectively several heuristic password-guessing algorithms guess passwords. Leveraging this method, we investigate (a) the resistance of passwords created under different conditions to password guessing; (b) the performance of guessing algorithms under different training sets; (c) the relationship between passwords explicitly created under a given composition policy and other passwords that happen to meet the same requirements; and (d) the relationship between guessability, as measured with password-cracking algorithms, and entropy estimates. We believe our findings advance understanding of both password-composition policies and metrics for quantifying password security.

Stephan Kroger - One of the best experts on this subject based on the ideXlab platform.

  • alternative splicing and the intracellular domain mediate tm Agrin s ability to differentially regulate the density of excitatory and inhibitory synapse like specializations in developing cns neurons
    Neuroscience, 2019
    Co-Authors: Gerry Handara, Stephan Kroger
    Abstract:

    Agrin is a multi-domain protein best known for its essential function during formation of the neuromuscular junction. Alternative mRNA splicing at sites named y and z in the C-terminal part of Agrin regulates its interaction with a receptor complex consisting of the Agrin-binding low-density lipoprotein receptor-related protein 4 (Lrp4) and the muscle-specific kinase (MuSK). Isoforms with inserts at both splice sites bind to Lrp4, activate MuSK and are synaptogenic at the neuromuscular junction. Agrin is also expressed as a transmembrane protein in the central nervous system (CNS) but its function during interneuronal synapse formation is unclear. Recently we demonstrated that transfection of a full-length cDNA coding for transmembrane Agrin (TM-Agrin) in cultured embryonic cortical neurons induced an Lrp4-dependent but MuSK-independent increase in dendritic glutamatergic synapses and an Lrp4- and MuSK-independent reduction of inhibitory synapses. Here we show that presynaptic specializations were similarly affected by TM-Agrin overexpression. In addition, we mapped the regions within TM-Agrin responsible for TM-Agrin's effects on dendritic aggregates of synapse-associated proteins. We show that the presence of a four amino acid insert at splice site y is essential for the increase in the density of puncta containing the postsynaptic density protein 95 kDa. This effect was independent of splice site z. The reduction of the gephyrin puncta density was independent of the entire extracellular part of TM-Agrin but required a highly conserved serine residue in the intracellular domain of TM-Agrin. These results provide further evidence for a function of TM-Agrin during CNS synaptogenesis and demonstrate that different domains and alternative splicing of TM-Agrin differentially affect excitatory and inhibitory synapse formation in cultured embryonic CNS neurons.

  • anti Agrin autoantibodies in myasthenia gravis
    Neurology, 2014
    Co-Authors: Christiane Gasperi, Arthur Melms, Benedikt Schoser, Yina Zhang, Julia Meltoranta, Valerie Risson, Laurent Schaeffer, Bertold Schalke, Stephan Kroger
    Abstract:

    Objective: Because the extracellular matrix protein Agrin is essential for neuromuscular junction formation and maintenance, we tested the hypothesis that autoantibodies against Agrin are present in sera from patients with myasthenia gravis (MG). Methods: We determined the presence of anti-Agrin antibodies in 54 sera from patients with generalized MG using a solid-phase ELISA with purified mini-Agrin protein. Thirty of the 54 sera were seronegative for antibodies against the acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK), 15 had elevated levels of anti-MuSK, and 9 had elevated levels of anti-AChR autoantibodies. Sixteen sera from healthy volunteers served as control. Results: Five sera with elevated levels of anti-Agrin antibodies were identified. The concentration of the antibodies ranged between 0.04 and 0.12 nM. Four of the 5 Agrin-positive sera were also positive for anti-MuSK, one was positive for anti-AChR, and 2 had elevated levels of anti–low-density lipoprotein receptor-related protein 4 (LRP4) autoantibodies. Some of the sera stained adult mouse neuromuscular junctions and reacted with native mini-Agrin expressed in 293HEK cells. Conclusions: The results provide evidence for Agrin as a novel target protein for autoantibodies in patients with MG. Anti-Agrin antibodies were always detected in combination with autoantibodies against MuSK, LRP4, or AChRs, indicating a high incidence of autoantibodies against several neuromuscular proteins in the Agrin-positive MG cases.

  • Agrin is a heparan sulfate proteoglycan
    Journal of Biological Chemistry, 1995
    Co-Authors: Guoshan Tsen, Willi Halfter, Stephan Kroger, Gregory J Cole
    Abstract:

    In the present study we have identified the extracellular matrix protein Agrin as a major heparan sulfate proteoglycan (HSPG) in embryonic chick brain. Using monoclonal antibodies and a polyclonal antiserum to the core protein of a previously identified HSPG from embryonic chick brain, our expression screened a random-primed E9 chick brain cDNA library. Twelve cDNAs were isolated that were shown to be identical to the chick extracellular matrix protein Agrin. Western blot analysis and immunocytochemistry confirmed that Agrin is a HSPG that is identical with the HSPG from embryonic chick brain. A polyclonal antiserum to recombinant Agrin protein recognized Agrin as a diffuse band of over 400 kDa in extracts from brain and vitreous humor. The Agrin immunoreactivity on the blot was shifted to a defined band of approximately 250 kDa after treatment of the samples with heparitinase or nitrous acid, and this banding pattern was indistinguishable from immunoreactivity obtained with antibodies to the brain HSPG. We also show that Agrin binds tightly to anion exchange beads, indicating that the molecule is highly negatively charged, which is a hallmark of all proteoglycans. Furthermore, the Agrin antiserum recognizes the affinity purified HSPG from chick brain and vitreous humor. Immunocytochemistry demonstrated that Agrin is expressed in developing brain, and is especially abundant in developing axonal tracts, in a distribution identical to the staining of the brain HSPG with monoclonal antibodies. We also show that the anti-HSPG antibodies stain the synaptic site of the neuromuscular junction, in agreement with Agrin expression. Thus, our studies demonstrate that chick Agrin is a HSPG that is prominent in the embryonic chick brain. Since previous studies from our laboratories have shown that this proteoglycan interacts with neural cell adhesion molecule, our studies raise the interesting possibility that neural cell adhesion molecule and Agrin are interactive partners that may regulate a variety of cell adhesion processes during neural development, including synaptogenesis.

Beatriz F. Fernandez - One of the best experts on this subject based on the ideXlab platform.

  • Agrin induces long-term osteochondral regeneration by supporting repair morphogenesis
    Science translational medicine, 2020
    Co-Authors: Suzanne E. Eldridge, Aida Barawi, Hui Wang, Anke J. Roelofs, Magdalena Kaneva, Zeyu Guan, Helen Lydon, B.l. Thomas, Anne-sophie Thorup, Beatriz F. Fernandez
    Abstract:

    Cartilage loss leads to osteoarthritis, the most common cause of disability for which there is no cure. Cartilage regeneration, therefore, is a priority in medicine. We report that Agrin is a potent chondrogenic factor and that a single intraarticular administration of Agrin induced long-lasting regeneration of critical-size osteochondral defects in mice, with restoration of tissue architecture and bone-cartilage interface. Agrin attracted joint resident progenitor cells to the site of injury and, through simultaneous activation of CREB and suppression of canonical WNT signaling downstream of β-catenin, induced expression of the chondrogenic stem cell marker GDF5 and differentiation into stable articular chondrocytes, forming stable articular cartilage. In sheep, an Agrin-containing collagen gel resulted in long-lasting regeneration of bone and cartilage, which promoted increased ambulatory activity. Our findings support the therapeutic use of Agrin for joint surface regeneration.

Hakima Moukhles - One of the best experts on this subject based on the ideXlab platform.

  • Agrin plays a major role in the coalescence of the aquaporin 4 clusters induced by gamma 1 containing laminin
    The Journal of Comparative Neurology, 2020
    Co-Authors: Geoffroy Noel, Daniel Kai Long Tham, Brian A Macvicar, Hakima Moukhles
    Abstract:

    The basement membrane that seperates the endothelial cells and astrocytic endfeet that comprise the blood-brain barrier is rich in collagen, laminin, Agrin, and perlecan. Previous studies have demonstrated that the proper recruitment of the water-permeable channel aquaporin-4 (AQP4) to astrocytic endfeet is dependent on interactions between laminin and the receptor dystroglycan. In this study, we conducted a deeper investigation into how the basement membrane might further regulate the expression, localization, and function of AQP4, using primary astrocytes as a model system. We found that treating these cells with laminin causes endogenous Agrin to localize to the cell surface, where it co-clusters with β-dystroglycan (β-DG). Conversely, Agrin sliencing profoundly disrupts β-DG clustering. As in the case of laminin111, Matrigel™, a complete basement membrane analog, also causes the clustering of AQP4 and β-DG. This clustering, whether induced by laminin111 or Matrigel™ is inhibited when the astrocytes are first incubated with an antibody against the γ1 subunit of laminin, suggesting that the latter is crucial to the process. Finally, we showed that laminin111 appears to negatively regulate AQP4-mediated water transport in astrocytes, suppressing the cell swelling that occurs following a hypoosmotic challenge. This suppression is abolished if DG expression is silenced, again demonstrating the central role of this receptor in relaying the effects of laminin.

  • dystroglycan and kir4 1 coclustering in retinal muller glia is regulated by laminin 1 and requires the pdz ligand domain of kir4 1
    Journal of Neurochemistry, 2005
    Co-Authors: Geoffrey Noel, Marina Belda, Eric Guadagno, Julien Micoud, Nikolaj Klocker, Hakima Moukhles
    Abstract:

    Inwardly rectifying potassium (Kir) channels in Muller glia play a critical role in the spatial buffering of potassium ions that accumulate during retinal activity. To this end, Kir channels show a polarized subcellular distribution with the predominant channel subunit in Muller glia, Kir4.1, clustered in the endfeet of these cells at the inner limiting membrane. However, the molecular mechanisms underlying their distribution have yet to be identified. Here, we show that laminin, Agrin and α-dystroglycan (DG) codistribute with Kir4.1 at the inner limiting membrane in the retina and that laminin-1 induces the clustering of α-DG, syntrophin and Kir4.1 in Muller cell cultures. In addition, we found that α-DG clusters were enriched for Agrin and sought to investigate the role of Agrin in their formation using recombinant C-Agrins. Both C-Agrin 4,8 and C-Agrin 0,0 failed to induce α-DG clustering and neither of them potentiated the α-DG clustering induced by laminin-1. Finally, our data reveal that deletion of the PDZ-ligand domain of Kir4.1 prevents their laminin-induced clustering. These findings indicate that both laminin-1 and α-DG are involved in the distribution of Kir4.1 to specific Muller cell membrane domains and that this process occurs via a PDZ-domain-mediated interaction. Thus, in the basal lamina laminin is an essential regulator involved in clearing excess potassium released during neuronal activity, thereby contributing to the maintenance of normal synaptic transmission in the retina.