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Kidney International Feb 2000Heparan sulfate (HS) is the anionic polysaccharide side chain of HS proteoglycans (HSPGs) present in basement membranes, in extracellular matrix, and on cell surfaces.... (Review)
Review
Heparan sulfate (HS) is the anionic polysaccharide side chain of HS proteoglycans (HSPGs) present in basement membranes, in extracellular matrix, and on cell surfaces. Recently, agrin was identified as a major HSPG present in the glomerular basement membrane (GBM). An increased permeability of the GBM for proteins after digestion of HS by heparitinase or after antibody binding to HS demonstrated the importance of HS for the permselective properties of the GBM. With recently developed antibodies directed against the GBM HSPG (agrin) core protein and the HS side chain, we demonstrated a decrease in HS staining in the GBM in different human proteinuric glomerulopathies, such as systemic lupus erythematosus (SLE), minimal change disease, membranous glomerulonephritis, and diabetic nephropathy, whereas the staining of the agrin core protein remained unaltered. This suggested changes in the HS side chains of HSPG in proteinuric glomerular diseases. To gain more insight into the mechanisms responsible for this observation, we studied GBM HS(PG) expression in experimental models of proteinuria. Similar HS changes were found in murine lupus nephritis, adriamycin nephropathy, and active Heymann nephritis. In these models, an inverse correlation was found between HS staining in the GBM and proteinuria. From these investigations, four new and different mechanisms have emerged. First, in lupus nephritis, HS was found to be masked by nucleosomes complexed to antinuclear autoantibodies. This masking was due to the binding of cationic moieties on the N-terminal parts of the core histones to anionic determinants in HS. Second, in adriamycin nephropathy, glomerular HS was depolymerized by reactive oxygen species (ROS), mainly hydroxyl radicals, which could be prevented by scavengers both in vitro (exposure of HS to ROS) and in vivo. Third, in vivo renal perfusion of purified elastase led to a decrease of HS in the GBM caused by proteolytic cleavage of the agrin core protein near the attachment sites of HS by the HS-bound enzyme. Fourth, in streptozotocin-induced diabetic nephropathy and during culture of glomerular cells under high glucose conditions, evidence was obtained that hyperglycemia led to a down-regulation of HS synthesis, accompanied by a reduction in the degree of HS sulfation.
Topics: Animals; Heparitin Sulfate; Humans; Kidney Glomerulus; Proteinuria
PubMed: 10652015
DOI: 10.1046/j.1523-1755.2000.00858.x -
Cellular and Molecular Life Sciences :... Sep 2013The neuromuscular junction (NMJ) is the most extensively studied model of neuronal synaptogenesis. Acetylcholine receptor (AChR) clustering on the postsynaptic membrane... (Review)
Review
The neuromuscular junction (NMJ) is the most extensively studied model of neuronal synaptogenesis. Acetylcholine receptor (AChR) clustering on the postsynaptic membrane is a cardinal event in the differentiation of NMJs. AChR clustering and postsynaptic differentiation is orchestrated by sophisticated interactions among three proteins: the neuron-secreted proteoglycan agrin, the co-receptor LRP4, and the muscle-specific receptor tyrosine kinase MuSK. LRP4 and MuSK act as scaffolds for multiple binding partners, resulting in a complex and dynamic network of interacting proteins that is required for AChR clustering. In this review, we discuss the structural basis for NMJ postsynaptic differentiation mediated by the agrin-LRP4-MuSK signaling pathway.
Topics: Agrin; Cell Differentiation; LDL-Receptor Related Proteins; Neuromuscular Junction; Receptors, Cholinergic; Signal Transduction
PubMed: 23178848
DOI: 10.1007/s00018-012-1209-9 -
PloS One 2014To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ)...
To determine if patients with myasthenia gravis (MG) have antibodies to agrin, a proteoglycan released by motor neurons and is critical for neuromuscular junction (NMJ) formation, we collected serum samples from 93 patients with MG with known status of antibodies to acetylcholine receptor (AChR), muscle specific kinase (MuSK) and lipoprotein-related 4 (LRP4) and samples from control subjects (healthy individuals and individuals with other diseases). Sera were assayed for antibodies to agrin. We found antibodies to agrin in 7 serum samples of MG patients. None of the 25 healthy controls and none of the 55 control neurological patients had agrin antibodies. Two of the four triple negative MG patients (i.e., no detectable AChR, MuSK or LRP4 antibodies, AChR-/MuSK-/LRP4-) had antibodies against agrin. In addition, agrin antibodies were detected in 5 out of 83 AChR+/MuSK-/LRP4- patients but were not found in the 6 patients with MuSK antibodies (AChR-/MuSK+/LRP4-). Sera from MG patients with agrin antibodies were able to recognize recombinant agrin in conditioned media and in transfected HEK293 cells. These sera also inhibited the agrin-induced MuSK phosphorylation and AChR clustering in muscle cells. Together, these observations indicate that agrin is another autoantigen in patients with MG and agrin autoantibodies may be pathogenic through inhibition of agrin/LRP4/MuSK signaling at the NMJ.
Topics: Agrin; Autoantibodies; Autoantigens; Humans; Myasthenia Gravis; Phosphorylation; Protein Binding; Receptor Protein-Tyrosine Kinases; Receptors, Cholinergic
PubMed: 24632822
DOI: 10.1371/journal.pone.0091816 -
PloS One May 2010The C-terminal 95 kDa fragment of some isoforms of vertebrate agrins is sufficient to induce clustering of acetylcholine receptors but despite two decades of intense...
The C-terminal 95 kDa fragment of some isoforms of vertebrate agrins is sufficient to induce clustering of acetylcholine receptors but despite two decades of intense agrin research very little is known about the function of the other isoforms and the function of the larger, N-terminal part of agrins that is common to all isoforms. Since the N-terminal part of agrins contains several follistatin-domains, a domain type that is frequently implicated in binding TGFbetas, we have explored the interaction of the N-terminal part of rat agrin (Agrin-Nterm) with members of the TGFbeta family using surface plasmon resonance spectroscopy and reporter assays. Here we show that agrin binds BMP2, BMP4 and TGFbeta1 with relatively high affinity, the K(D) values of the interactions calculated from SPR experiments fall in the 10(-8) M-10(-7) M range. In reporter assays Agrin-Nterm inhibited the activities of BMP2 and BMP4, half maximal inhibition being achieved at approximately 5x10(-7) M. Paradoxically, in the case of TGFbeta1 Agrin N-term caused a slight increase in activity in reporter assays. Our finding that agrin binds members of the TGFbeta family may have important implications for the role of these growth factors in the regulation of synaptogenesis as well as for the role of agrin isoforms that are unable to induce clustering of acetylcholine receptors. We suggest that binding of these TGFbeta family members to agrin may have a dual function: agrin may serve as a reservoir for these growth factors and may also inhibit their growth promoting activity. Based on analysis of the evolutionary history of agrin we suggest that agrin's growth factor binding function is more ancient than its involvement in acetylcholine receptor clustering.
Topics: Agrin; Animals; Bone Morphogenetic Protein 2; Bone Morphogenetic Protein 4; Bone Morphogenetic Protein Receptors; Cell Line; Chromatography, Affinity; Chromatography, Gel; Evolution, Molecular; Humans; Intercellular Signaling Peptides and Proteins; Kinetics; Protein Binding; Protein Structure, Tertiary; Rats; Receptors, Transforming Growth Factor beta; Surface Plasmon Resonance; Transforming Growth Factor beta1
PubMed: 20505824
DOI: 10.1371/journal.pone.0010758 -
Trends in Neurosciences Jan 1998The 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... (Review)
Review
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.
Topics: Agrin; Animals; Cell Differentiation; Humans; Neuromuscular Junction; Neurons; Synapses
PubMed: 9464682
DOI: 10.1016/s0166-2236(97)01154-5 -
Molecular Biology of the Cell Sep 2019Key genes, such as , , and , are required for the initial formation, subsequent maturation, and long-term stabilization of mammalian neuromuscular synapses. Additional...
Key genes, such as , , and , are required for the initial formation, subsequent maturation, and long-term stabilization of mammalian neuromuscular synapses. Additional molecules are thought to function selectively during the evolution and stabilization of these synapses, but these molecular players are largely unknown. Here, we used mass spectrometry to identify vezatin, a two-pass transmembrane protein, as an acetylcholine receptor (AChR)-associated protein, and we provide evidence that vezatin binds directly to AChRs. We show that vezatin is dispensable for the formation of synapses but plays a later role in the emergence of a topologically complex and branched shape of the synapse, as well as the stabilization of AChRs. In addition, neuromuscular synapses in mutant mice display premature signs of deterioration, normally found only during aging. Thus, vezatin has a selective role in the structural elaboration and postnatal maturation of murine neuromuscular synapses.
Topics: Agrin; Animals; Carrier Proteins; Cell Line; Membrane Proteins; Mice; Mice, Knockout; Muscle Proteins; Neuromuscular Junction; Phosphorylation; Receptors, Nicotinic; Synapses
PubMed: 31411944
DOI: 10.1091/mbc.E19-06-0313 -
Cell Reports Mar 2017The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of...
The Hippo pathway effectors YAP and TAZ act as nuclear sensors of mechanical signals in response to extracellular matrix (ECM) cues. However, the identity and nature of regulators in the ECM and the precise pathways relaying mechanoresponsive signals into intracellular sensors remain unclear. Here, we uncover a functional link between the ECM proteoglycan Agrin and the transcriptional co-activator YAP. Importantly, Agrin transduces matrix and cellular rigidity signals that enhance stability and mechanoactivity of YAP through the integrin-focal adhesion- and Lrp4/MuSK receptor-mediated signaling pathways. Agrin antagonizes focal adhesion assembly of the core Hippo components by facilitating ILK-PAK1 signaling and negating the functions of Merlin and LATS1/2. We further show that Agrin promotes oncogenesis through YAP-dependent transcription and is clinically relevant in human liver cancer. We propose that Agrin acts as a mechanotransduction signal in the ECM.
Topics: 14-3-3 Proteins; Adaptor Proteins, Signal Transducing; Agrin; Animals; Carcinogenesis; Cell Line; Cell Line, Tumor; Cytoskeleton; Extracellular Matrix; Female; Focal Adhesion Protein-Tyrosine Kinases; Focal Adhesions; Humans; Mechanotransduction, Cellular; Mice, Nude; Phosphoproteins; Protein Binding; Protein Stability; Signal Transduction; Transcription Factors; YAP-Signaling Proteins
PubMed: 28273460
DOI: 10.1016/j.celrep.2017.02.041 -
The Journal of Neuroscience : the... Oct 2018During aging, acetylcholine receptor (AChR) clusters become fragmented and denervated at the neuromuscular junction (NMJ). Underpinning molecular mechanisms are not well...
During aging, acetylcholine receptor (AChR) clusters become fragmented and denervated at the neuromuscular junction (NMJ). Underpinning molecular mechanisms are not well understood. We showed that LRP4, a receptor for agrin and critical for NMJ formation and maintenance, was reduced at protein level in aged mice, which was associated with decreased MuSK tyrosine phosphorylation, suggesting compromised agrin-LRP4-MuSK signaling in aged muscles. Transgenic expression of LRP4 in muscles alleviated AChR fragmentation and denervation and improved neuromuscular transmission in aged mice. LRP4 ubiquitination was augmented in aged muscles, suggesting increased LRP4 degradation as a mechanism for reduced LRP4. We found that sarcoglycan α (SGα) interacted with LRP4 and delayed LRP4 degradation in cotransfected cells. AAV9-mediated expression of SGα in muscles mitigated AChR fragmentation and denervation and improved neuromuscular transmission in aged mice. These observations support a model where compromised agrin-LRP4-MuSK signaling serves as a pathological mechanism of age-related NMJ decline and identify a novel function of SGα in stabilizing LRP4 for NMJ stability in aged mice. This study provides evidence that LRP4, a receptor of agrin that is critical for NMJ formation and maintenance, is reduced at protein level in aged muscles. Transgenic expression of LRP4 in muscles ameliorates AChR fragmentation and denervation and improves neuromuscular transmission in aged mice, demonstrating a critical role of the agrin-LRP4-MuSK signaling. Our study also reveals a novel function of SGα to prevent LRP4 degradation in aged muscles. Finally, we show that NMJ decline in aged mice can be mitigated by AAV9-mediated expression of SGα in muscles. These observations provide insight into pathological mechanisms of age-related NMJ decline and suggest that improved agrin-LRP4-MuSK signaling may be a target for potential therapeutic intervention.
Topics: Aging; Animals; Female; LDL-Receptor Related Proteins; Male; Mice, Inbred C57BL; Mice, Transgenic; Muscle, Skeletal; Neuromuscular Junction; Phosphorylation; Receptor Protein-Tyrosine Kinases; Receptors, Cholinergic; Receptors, LDL; Sarcoglycans
PubMed: 30171091
DOI: 10.1523/JNEUROSCI.0860-18.2018 -
Current Opinion in Physiology Aug 2018As the final output of the somatic nervous system, the neuromuscular junction (NMJ) is essential for all voluntary movements. The NMJ is also necessary for connected...
As the final output of the somatic nervous system, the neuromuscular junction (NMJ) is essential for all voluntary movements. The NMJ is also necessary for connected cells to function and survive. Because of this central role, much effort has been devoted to understanding the effects of aging, diseases, and injuries on the NMJ. These efforts have revealed a close relationship between aberrant changes at NMJs and its three cellular components - the presynaptic site on motor axons, the postsynaptic region on muscle fibers and perisynaptic Schwann cells. Here, we review the morphological and molecular changes associated with aging NMJs in rodents and humans. We also provide an overview of factors with potential roles in maintaining and repairing adult and aged NMJs.
PubMed: 30560223
DOI: 10.1016/j.cophys.2018.05.007 -
The Journal of Neuroscience : the... Oct 2014The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires...
The neuromuscular junction (NMJ) is a synapse between motor neurons and skeletal muscle fibers, and is critical for control of muscle contraction. Its formation requires neuronal agrin that acts by binding to LRP4 to stimulate MuSK. Mutations have been identified in agrin, MuSK, and LRP4 in patients with congenital myasthenic syndrome, and patients with myasthenia gravis develop antibodies against agrin, LRP4, and MuSK. However, it remains unclear whether the agrin signaling pathway is critical for NMJ maintenance because null mutation of any of the three genes is perinatal lethal. In this study, we generated imKO mice, a mutant strain whose LRP4 gene can be deleted in muscles by doxycycline (Dox) treatment. Ablation of the LRP4 gene in adult muscle enabled studies of its role in NMJ maintenance. We demonstrate that Dox treatment of P30 mice reduced muscle strength and compound muscle action potentials. AChR clusters became fragmented with diminished junctional folds and synaptic vesicles. The amplitude and frequency of miniature endplate potentials were reduced, indicating impaired neuromuscular transmission and providing cellular mechanisms of adult LRP4 deficiency. We showed that LRP4 ablation led to the loss of synaptic agrin and the 90 kDa fragments, which occurred ahead of other prejunctional and postjunctional components, suggesting that LRP4 may regulate the stability of synaptic agrin. These observations demonstrate that LRP4 is essential for maintaining the structural and functional integrity of the NMJ and that loss of muscle LRP4 in adulthood alone is sufficient to cause myasthenic symptoms.
Topics: Animals; Humans; LDL-Receptor Related Proteins; Male; Mice; Mice, Inbred C57BL; Mice, Knockout; Mice, Transgenic; Neuromuscular Junction; Receptors, LDL
PubMed: 25319686
DOI: 10.1523/JNEUROSCI.1733-14.2014