RESUMEN
Congenital myasthenia (CM) is a devastating neuromuscular disease, and mutations in DOK7, an adaptor protein that is crucial for forming and maintaining neuromuscular synapses, are a major cause of CM1,2. The most common disease-causing mutation (DOK71124_1127 dup) truncates DOK7 and leads to the loss of two tyrosine residues that are phosphorylated and recruit CRK proteins, which are important for anchoring acetylcholine receptors at synapses. Here we describe a mouse model of this common form of CM (Dok7CM mice) and a mouse with point mutations in the two tyrosine residues (Dok72YF). We show that Dok7CM mice had severe deficits in neuromuscular synapse formation that caused neonatal lethality. Unexpectedly, these deficits were due to a severe deficiency in phosphorylation and activation of muscle-specific kinase (MUSK) rather than a deficiency in DOK7 tyrosine phosphorylation. We developed agonist antibodies against MUSK and show that these antibodies restored neuromuscular synapse formation and prevented neonatal lethality and late-onset disease in Dok7CM mice. These findings identify an unexpected cause for disease and a potential therapy for both DOK7 CM and other forms of CM caused by mutations in AGRIN, LRP4 or MUSK, and illustrate the potential of targeted therapy to rescue congenital lethality.
Asunto(s)
Proteínas Musculares/genética , Mutación , Síndromes Miasténicos Congénitos/tratamiento farmacológico , Síndromes Miasténicos Congénitos/genética , Envejecimiento , Agrina/genética , Agrina/metabolismo , Animales , Animales Recién Nacidos , Anticuerpos/inmunología , Modelos Animales de Enfermedad , Femenino , Proteínas Relacionadas con Receptor de LDL/genética , Proteínas Relacionadas con Receptor de LDL/metabolismo , Masculino , Ratones , Terapia Molecular Dirigida , Fibras Musculares Esqueléticas/química , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/química , Proteínas Musculares/metabolismo , Síndromes Miasténicos Congénitos/inmunología , Fosforilación , Fosfotirosina/genética , Fosfotirosina/metabolismo , Proteínas Proto-Oncogénicas c-crk/metabolismo , Proteínas Tirosina Quinasas Receptoras/agonistas , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/inmunología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Recurrencia , Sinapsis/metabolismoRESUMEN
Aptamers with fluorogenic ligands are emerging as useful tools to quantify and track RNA molecules. The RNA Mango family of aptamers have a useful combination of tight ligand binding, bright fluorescence, and small size. However, the simple structure of these aptamers, with a single base-paired stem capped by a G-quadruplex, can limit the sequence and structural modifications needed for many use-inspired designs. Here we report new structural variants of RNA Mango that have two base-paired stems attached to the quadruplex. Fluorescence saturation analysis of one of the double-stemmed constructs showed a maximum fluorescence that is â¼75% brighter than the original single-stemmed Mango I. A small number of mutations to nucleotides in the tetraloop-like linker of the second stem were subsequently analyzed. The effect of these mutations on the affinity and fluorescence suggested that the nucleobases of the second linker do not directly interact with the fluorogenic ligand (TO1-biotin), but may instead induce higher fluorescence by indirectly altering the ligand properties in the bound state. The effects of the mutations in this second tetraloop-like linker indicate the potential of this second stem for rational design and reselection experiments. Additionally, we demonstrated that a bimolecular mango designed by splitting the double-stemmed Mango can function when two RNA molecules are cotranscribed from different DNA templates in a single in vitro transcription. This bimolecular Mango has potential application in detecting RNA-RNA interactions. Together, these constructs expand the designability of the Mango aptamers to facilitate future applications of RNA imaging.
Asunto(s)
Aptámeros de Nucleótidos , Mangifera , Mangifera/genética , Mangifera/química , Mangifera/metabolismo , Aptámeros de Nucleótidos/química , Ligandos , Colorantes Fluorescentes/química , ARN/químicaRESUMEN
Motor axons approach muscles that are prepatterned in the prospective synaptic region. In mice, prepatterning of acetylcholine receptors requires Lrp4, a LDLR family member, and MuSK, a receptor tyrosine kinase. Lrp4 can bind and stimulate MuSK, strongly suggesting that association between Lrp4 and MuSK, independent of additional ligands, initiates prepatterning in mice. In zebrafish, Wnts, which bind the Frizzled (Fz)-like domain in MuSK, are required for prepatterning, suggesting that Wnts may contribute to prepatterning and neuromuscular development in mammals. We show that prepatterning in mice requires Lrp4 but not the MuSK Fz-like domain. In contrast, prepatterning in zebrafish requires the MuSK Fz-like domain but not Lrp4. Despite these differences, neuromuscular synapse formation in zebrafish and mice share similar mechanisms, requiring Lrp4, MuSK, and neuronal Agrin but not the MuSK Fz-like domain or Wnt production from muscle. Our findings demonstrate that evolutionary divergent mechanisms establish muscle prepatterning in zebrafish and mice.
Asunto(s)
Evolución Biológica , Proteínas de la Matriz Extracelular/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Unión Neuromuscular/embriología , Unión Neuromuscular/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Tipificación del Cuerpo/genética , Proteínas de la Matriz Extracelular/genética , Proteínas de Homeodominio , Ratones , Proteínas del Tejido Nervioso/genética , Proteínas Tirosina Quinasas Receptoras/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Neuromuscular synapse formation requires a complex exchange of signals between motor neurons and skeletal muscle fibers, leading to the accumulation of postsynaptic proteins, including acetylcholine receptors in the muscle membrane and specialized release sites, or active zones in the presynaptic nerve terminal. MuSK, a receptor tyrosine kinase that is expressed in skeletal muscle, and Agrin, a motor neuron-derived ligand that stimulates MuSK phosphorylation, play critical roles in synaptic differentiation, as synapses do not form in their absence, and mutations in MuSK or downstream effectors are a major cause of a group of neuromuscular disorders, termed congenital myasthenic syndromes (CMS). How Agrin activates MuSK and stimulates synaptic differentiation is not known and remains a fundamental gap in our understanding of signaling at neuromuscular synapses. Here, we report that Lrp4, a member of the LDLR family, is a receptor for Agrin, forms a complex with MuSK, and mediates MuSK activation by Agrin.
Asunto(s)
Agrina/metabolismo , Unión Neuromuscular/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores de LDL/metabolismo , Animales , Línea Celular , Proteínas Relacionadas con Receptor de LDL , Ratones , Modelos Biológicos , Mioblastos/metabolismo , Fosforilación , Células Precursoras de Linfocitos B/metabolismoRESUMEN
The neural cell adhesion molecule (NCAM) is expressed both presynaptically and postsynaptically during neuromuscular junction formation. Genetic deletion in mice of all three isoforms (180, 140, and 120 kDa), or just the 180 isoform, suggested that different isoforms played distinct roles in synaptic maturation. Here we characterized in mice of either sex the earliest adhesive contacts between the growth cones of motoneurons and myotubes and their subsequent maturation into functional synapses in cocultures of motoneurons and myotubes, which expressed their normal complement of NCAM isoforms, or were lacking all isoforms either presynaptically or postsynaptically. Growth cone contact with +/+ mouse myotubes resulted in immediate adhesive contacts and the rapid downregulation of growth cone motility. When contacting NCAM-/- myotubes, growth cones touched and retracted/collapsed multiple times and failed to form stable contacts, even after 10 h. Exogenous expression in myotubes of either the 180 or 140 isoform, but not the 120 kDa isoform, rescued the rapid formation of stable contacts, the accumulation of presynaptic and postsynaptic molecules, and functional transmission. When NCAM was absent only in motoneurons, growth cones did not retract upon myotube contact, but, since their motility was not downregulated, they grew off the ends of the myotubes, failing to form synapses. The agrin receptor Lrp4 was strongly downregulated in NCAM-negative myotubes, and motoneuron growth cones did not make stable contacts with Lrp4-negative myotubes. These studies have identified novel roles for presynaptic and postsynaptic NCAM in mediating early cell-cell interactions required for synapse formation.SIGNIFICANCE STATEMENT Although many molecular signals needed to form the functionally effective neuromuscular synapses required for normal movement have been described, the earliest signals that let motoneuron growth cones make stable adhesive contacts with myotubes and cease motility are not well understood. Using dynamic imaging of motoneuron-myotube cocultures, we show that NCAM is required on both the growth cone and myotube and that different NCAM isoforms mediate initial adhesion and the downregulation of growth cone motility. The agrin receptor Lrp4 was also essential for initial adhesive contacts and was downregulated on NCAM-/- myotubes. Our identification of novel roles for NCAM and Lrp4 and possible interactions between them in transforming motile growth cones into stable contacts opens interesting new avenues for exploration.
Asunto(s)
Conos de Crecimiento/metabolismo , Neuronas Motoras/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Moléculas de Adhesión de Célula Nerviosa/metabolismo , Neurogénesis/fisiología , Sinapsis/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Unión Neuromuscular/crecimiento & desarrollo , Unión Neuromuscular/metabolismo , Isoformas de ProteínasRESUMEN
Motor axons receive retrograde signals from skeletal muscle that are essential for the differentiation and stabilization of motor nerve terminals. Identification of these retrograde signals has proved elusive, but their production by muscle depends on the receptor tyrosine kinase, MuSK (muscle, skeletal receptor tyrosine-protein kinase), and Lrp4 (low-density lipoprotein receptor (LDLR)-related protein 4), an LDLR family member that forms a complex with MuSK, binds neural agrin and stimulates MuSK kinase activity. Here we show that Lrp4 also functions as a direct muscle-derived retrograde signal for early steps in presynaptic differentiation. We demonstrate that Lrp4 is necessary, independent of MuSK activation, for presynaptic differentiation in vivo, and we show that Lrp4 binds to motor axons and induces clustering of synaptic-vesicle and active-zone proteins. Thus, Lrp4 acts bidirectionally and coordinates synapse formation by binding agrin, activating MuSK and stimulating postsynaptic differentiation, and functioning in turn as a muscle-derived retrograde signal that is necessary and sufficient for presynaptic differentiation.
Asunto(s)
Diferenciación Celular , Músculo Esquelético/inervación , Unión Neuromuscular/citología , Unión Neuromuscular/metabolismo , Terminales Presinápticos/metabolismo , Receptores de LDL/metabolismo , Transducción de Señal , Esclerosis Amiotrófica Lateral , Animales , Células Cultivadas , Técnicas de Cocultivo , Diafragma , Proteínas Relacionadas con Receptor de LDL , Ratones , Neuronas Motoras/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Tubo Neural/citología , Tubo Neural/metabolismo , Unión Proteica , Proteínas Tirosina Quinasas Receptoras/metabolismo , Sarcopenia , Sinapsinas/metabolismo , Técnicas de Cultivo de TejidosRESUMEN
Formation of the vertebrate neuromuscular junction requires, among others proteins, Agrin, a neuronally derived ligand, and the following muscle proteins: LRP4, the receptor for Agrin; MuSK, a receptor tyrosine kinase (RTK); and Dok7 (or Dok-7), a cytoplasmic adaptor protein. Dok7 comprises a pleckstrin-homology (PH) domain, a phosphotyrosine-binding (PTB) domain, and C-terminal sites of tyrosine phosphorylation. Unique among adaptor proteins recruited to RTKs, Dok7 is not only a substrate of MuSK, but also an activator of MuSK's kinase activity. Here, we present the crystal structure of the Dok7 PH-PTB domains in complex with a phosphopeptide representing the Dok7-binding site on MuSK. The structure and biochemical data reveal a dimeric arrangement of Dok7 PH-PTB that facilitates trans-autophosphorylation of the kinase activation loop. The structure provides the molecular basis for MuSK activation by Dok7 and for rationalizing several Dok7 loss-of-function mutations found in patients with congenital myasthenic syndromes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Citoplasma/metabolismo , Proteínas Musculares/metabolismo , Multimerización de Proteína , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Secuencia de Aminoácidos , Animales , Cristalografía por Rayos X , Activación Enzimática , Humanos , Ratones , Modelos Moleculares , Datos de Secuencia Molecular , Proteínas Musculares/química , Mutación/genética , Células 3T3 NIH , Fosfatidilinositoles/metabolismo , Fosforilación , Fosfotirosina/metabolismo , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Ratas , Proteínas Tirosina Quinasas Receptoras/química , Receptores Colinérgicos/químicaRESUMEN
Agrin, released by motor neurons, promotes neuromuscular synapse formation by stimulating MuSK, a receptor tyrosine kinase expressed in skeletal muscle. Phosphorylated MuSK recruits docking protein-7 (Dok-7), an adaptor protein that is expressed selectively in muscle. In the absence of Dok-7, neuromuscular synapses fail to form, and mutations that impair Dok-7 are a major cause of congenital myasthenia in humans. How Dok-7 stimulates synaptic differentiation is poorly understood. Once recruited to MuSK, Dok-7 directly stimulates MuSK kinase activity. This unusual activity of an adapter protein is mediated by the N-terminal region of Dok-7, whereas most mutations that cause congenital myasthenia truncate the C-terminal domain. Here, we demonstrate that Dok-7 also functions downstream from MuSK, and we identify the proteins that are recruited to the C-terminal domain of Dok-7. We show that Agrin stimulates phosphorylation of two tyrosine residues in the C-terminal domain of Dok-7, which leads to recruitment of two adapter proteins: Crk and Crk-L. Furthermore, we show that selective inactivation of Crk and Crk-L in skeletal muscle leads to severe defects in neuromuscular synapses in vivo, revealing a critical role for Crk and Crk-L downstream from Dok-7 in presynaptic and postsynaptic differentiation.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Musculares/metabolismo , Unión Neuromuscular/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogénicas c-crk/metabolismo , Sinapsis/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Agrina/farmacología , Animales , Western Blotting , Línea Celular , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Ratones , Ratones Noqueados , Microscopía Confocal , Fibras Musculares Esqueléticas/citología , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Músculo Esquelético/embriología , Músculo Esquelético/metabolismo , Mutación , Proteínas Nucleares/genética , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-crk/genética , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Factores de Tiempo , Tirosina/genética , Tirosina/metabolismoRESUMEN
The neuromuscular synapse is a relatively large synapse with hundreds of active zones in presynaptic motor nerve terminals and more than ten million acetylcholine receptors (AChRs) in the postsynaptic membrane. The enrichment of proteins in presynaptic and postsynaptic membranes ensures a rapid, robust, and reliable synaptic transmission. Over fifty years ago, classic studies of the neuromuscular synapse led to a comprehensive understanding of how a synapse looks and works, but these landmark studies did not reveal the molecular mechanisms responsible for building and maintaining a synapse. During the past two-dozen years, the critical molecular players, responsible for assembling the specialized postsynaptic membrane and regulating nerve terminal differentiation, have begun to be identified and their mechanism of action better understood. Here, we describe and discuss five of these key molecular players, paying heed to their discovery as well as describing their currently understood mechanisms of action. In addition, we discuss the important gaps that remain to better understand how these proteins act to control synaptic differentiation and maintenance.
Asunto(s)
Unión Neuromuscular/patología , Transmisión Sináptica , Animales , Biomarcadores , Humanos , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas Musculares/metabolismo , Receptores Colinérgicos/metabolismoRESUMEN
Myasthenia gravis (MG) is a severely debilitating autoimmune disease that is due to a decrease in the efficiency of synaptic transmission at neuromuscular synapses. MG is caused by antibodies against postsynaptic proteins, including (i) acetylcholine receptors, the neurotransmitter receptor, (ii) muscle-specific kinase (MuSK), a receptor tyrosine kinase essential for the formation and maintenance of neuromuscular synapses, and (iii) low-density lipoprotein receptor-related protein 4 (Lrp4), which responds to neural Agrin by binding and stimulating MuSK. Passive transfer studies in mice have shown that IgG4 antibodies from MuSK MG patients cause disease without requiring complement or other immune components, suggesting that these MuSK antibodies cause disease by directly interfering with MuSK function. Here we show that pathogenic IgG4 antibodies to MuSK bind to a structural epitope in the first Ig-like domain of MuSK, prevent binding between MuSK and Lrp4, and inhibit Agrin-stimulated MuSK phosphorylation. In contrast, these IgG4 antibodies have no direct effect on MuSK dimerization or MuSK internalization. These results provide insight into the unique pathogenesis of MuSK MG and provide clues toward development of specific treatment options.
Asunto(s)
Autoanticuerpos/inmunología , Inmunoglobulina G/inmunología , Proteínas Relacionadas con Receptor de LDL/inmunología , Miastenia Gravis/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores Colinérgicos/inmunología , Receptores de LDL/inmunología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Agrina/inmunología , Animales , Autoanticuerpos/farmacología , Línea Celular , Niño , Preescolar , Epítopos/inmunología , Femenino , Humanos , Inmunización Pasiva , Inmunoglobulina G/farmacología , Proteínas Relacionadas con Receptor de LDL/antagonistas & inhibidores , Masculino , Ratones , Persona de Mediana Edad , Miastenia Gravis/inducido químicamente , Miastenia Gravis/patología , Fosforilación/efectos de los fármacos , Fosforilación/inmunología , Multimerización de Proteína/efectos de los fármacos , Multimerización de Proteína/inmunología , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Receptores de LDL/antagonistas & inhibidoresRESUMEN
Dystroglycan, which serves as a major extracellular matrix receptor in muscle and the central nervous system, requires extensive O-glycosylation to function. We identified a dystroglycan missense mutation (Thr192âMet) in a woman with limb-girdle muscular dystrophy and cognitive impairment. A mouse model harboring this mutation recapitulates the immunohistochemical and neuromuscular abnormalities observed in the patient. In vitro and in vivo studies showed that the mutation impairs the receptor function of dystroglycan in skeletal muscle and brain by inhibiting the post-translational modification, mediated by the glycosyltransferase LARGE, of the phosphorylated O-mannosyl glycans on α-dystroglycan that is required for high-affinity binding to laminin.
Asunto(s)
Distroglicanos/genética , Distrofia Muscular de Cinturas/genética , Mutación Missense , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Linaje , Fenotipo , Análisis de Secuencia de ADNRESUMEN
Trunk neural crest cells delaminate from the dorsal neural tube as an uninterrupted sheet; however, they convert into segmentally organized streams before migrating through the somitic territory. These neural crest cell streams join the segmental trajectories of pathfinding spinal motor axons, suggesting that interactions between these two cell types might be important for neural crest cell migration. Here, we show that in the zebrafish embryo migration of both neural crest cells and motor axons is temporally synchronized and spatially restricted to the center of the somite, but that motor axons are dispensable for segmental neural crest cell migration. Instead, we find that muscle-specific receptor kinase (MuSK) and its putative ligand Wnt11r are crucial for restricting neural crest cell migration to the center of each somite. Moreover, we find that blocking planar cell polarity (PCP) signaling in somitic muscle cells also results in non-segmental neural crest cell migration. Using an F-actin biosensor we show that in the absence of MuSK neural crest cells fail to retract non-productive leading edges, resulting in non-segmental migration. Finally, we show that MuSK knockout mice display similar neural crest cell migration defects, suggesting a novel, evolutionarily conserved role for MuSK in neural crest migration. We propose that a Wnt11r-MuSK dependent, PCP-like pathway restricts neural crest cells to their segmental path.
Asunto(s)
Movimiento Celular/fisiología , Cresta Neural/citología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal/fisiología , Proteínas Wnt/metabolismo , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Animales , Proteínas de Homeodominio , Ratones , Ratones Noqueados , Morfogénesis/fisiología , Cresta Neural/fisiología , Proteínas Tirosina Quinasas Receptoras/genética , Proteínas Wnt/genética , Pez Cebra/anatomía & histología , Pez Cebra/genética , Pez Cebra/fisiología , Proteínas de Pez Cebra/genéticaRESUMEN
Agrin, an extracellular matrix protein belonging to the heterogeneous family of heparan sulfate proteoglycans (HSPGs), is expressed by cells of the hematopoietic system but its role in leukocyte biology is not yet clear. Here we demonstrate that agrin has a crucial, nonredundant role in myeloid cell development and functions. We have identified lineage-specific alterations that affect maturation, survival and properties of agrin-deficient monocytic cells, and occur at stages later than stem cell precursors. Our data indicate that the cell-autonomous signals delivered by agrin are sensed by macrophages through the α-DC (DG) receptor and lead to the activation of signaling pathways resulting in rearrangements of the actin cytoskeleton during the phagocytic synapse formation and phosphorylation of extracellular signal-regulated kinases (Erk 1/2). Altogether, these data identify agrin as a novel player of innate immunity.
Asunto(s)
Agrina/metabolismo , Células Mieloides/citología , Mielopoyesis , Agrina/análisis , Agrina/genética , Animales , Supervivencia Celular , Distroglicanos/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Macrófagos/citología , Macrófagos/metabolismo , Ratones , Ratones Endogámicos C57BL , Monocitos/citología , Monocitos/metabolismo , Células Mieloides/metabolismo , Fagocitosis , FosforilaciónRESUMEN
α-dystroglycan is a highly O-glycosylated extracellular matrix receptor that is required for anchoring of the basement membrane to the cell surface and for the entry of Old World arenaviruses into cells. Like-acetylglucosaminyltransferase (LARGE) is a key molecule that binds to the N-terminal domain of α-dystroglycan and attaches ligand-binding moieties to phosphorylated O-mannose on α-dystroglycan. Here we show that the LARGE modification required for laminin- and virus-binding occurs on specific Thr residues located at the extreme N terminus of the mucin-like domain of α-dystroglycan. Deletion and mutation analyses demonstrate that the ligand-binding activity of α-dystroglycan is conferred primarily by LARGE modification at Thr-317 and -319, within the highly conserved first 18 amino acids of the mucin-like domain. The importance of these paired residues in laminin-binding and clustering activity on myoblasts and in arenavirus cell entry is confirmed by mutational analysis with full-length dystroglycan. We further demonstrate that a sequence of five amino acids, Thr(317)ProThr(319)ProVal, contains phosphorylated O-glycosylation and, when modified by LARGE is sufficient for laminin-binding. Because the N-terminal region adjacent to the paired Thr residues is removed during posttranslational maturation of dystroglycan, our results demonstrate that the ligand-binding activity resides at the extreme N terminus of mature α-dystroglycan and is crucial for α-dystroglycan to coordinate the assembly of extracellular matrix proteins and to bind arenaviruses on the cell surface.
Asunto(s)
Infecciones por Arenaviridae/etiología , Infecciones por Arenaviridae/metabolismo , Distroglicanos/metabolismo , Laminina/metabolismo , Virus de la Coriomeningitis Linfocítica , N-Acetilglucosaminiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión , Línea Celular , Distroglicanos/química , Distroglicanos/genética , Glicosilación , Células HEK293 , Humanos , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Modelos Biológicos , Datos de Secuencia Molecular , Mutagénesis , Mioblastos/metabolismo , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina/química , Internalización del VirusRESUMEN
A coordinated and complex interplay of signals between motor neurons, skeletal muscle cells, and Schwann cells controls the formation and maintenance of neuromuscular synapses. Deficits in the signaling pathway for building synapses, caused by mutations in critical genes or autoantibodies against key proteins, are responsible for several neuromuscular diseases, which cause muscle weakness and fatigue. Here, we describe the role that four key genes, Agrin, Lrp4, MuSK, and Dok7, play in this signaling pathway, how an understanding of their mechanisms of action has led to an understanding of several neuromuscular diseases, and how this knowledge has contributed to emerging therapies for treating neuromuscular diseases.
Asunto(s)
Unión Neuromuscular , Transducción de Señal , Humanos , Animales , Agrina/metabolismo , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas Tirosina Quinasas Receptoras/metabolismo , Proteínas Musculares/metabolismo , Enfermedades Neuromusculares , Receptores Colinérgicos/metabolismo , Sinapsis/fisiología , Sinapsis/metabolismo , Neuronas Motoras/fisiología , Neuronas Motoras/metabolismoRESUMEN
Hematopoiesis is the process leading to the sustained production of blood cells by hematopoietic stem cells (HSCs). Growth, survival, and differentiation of HSCs occur in specialized microenvironments called "hematopoietic niches," through molecular cues that are only partially understood. Here we show that agrin, a proteoglycan involved in the neuromuscular junction, is a critical niche-derived signal that controls survival and proliferation of HSCs. Agrin is expressed by multipotent nonhematopoietic mesenchymal stem cells (MSCs) and by differentiated osteoblasts lining the endosteal bone surface, whereas Lin(-)Sca1(+)c-Kit(+) (LSK) cells express the α-dystroglycan receptor for agrin. In vitro, agrin-deficient MSCs were less efficient in supporting proliferation of mouse Lin(-)c-Kit(+) cells, suggesting that agrin plays a role in the hematopoietic cell development. These results were indeed confirmed in vivo through the analysis of agrin knockout mice (Musk-L;Agrn(-/-)). Agrin-deficient mice displayed in vivo apoptosis of CD34(+)CD135(-) LSK cells and impaired hematopoiesis, both of which were reverted by an agrin-sufficient stroma. These data unveil a crucial role of agrin in the hematopoietic niches and in the cross-talk between stromal and hematopoietic stem cells.
Asunto(s)
Agrina/fisiología , Proliferación Celular , Hematopoyesis , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/fisiología , Nicho de Células Madre , Animales , Western Blotting , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Células Cultivadas , Femenino , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Técnicas para Inmunoenzimas , Masculino , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Osteoblastos/citología , Osteoblastos/metabolismo , ARN Mensajero/genética , Receptores de Factores de Crecimiento , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Autophagy allows cell survival during starvation through the bulk degradation of proteins and organelles by lysosomal enzymes. However, the mechanisms responsible for the induction and regulation of the autophagy program are poorly understood. Here we show that the FoxO3 transcription factor, which plays a critical role in muscle atrophy, is necessary and sufficient for the induction of autophagy in skeletal muscle in vivo. Akt/PKB activation blocks FoxO3 activation and autophagy, and this effect is not prevented by rapamycin. FoxO3 controls the transcription of autophagy-related genes, including LC3 and Bnip3, and Bnip3 appears to mediate the effect of FoxO3 on autophagy. This effect is not prevented by proteasome inhibitors. Thus, FoxO3 controls the two major systems of protein breakdown in skeletal muscle, the ubiquitin-proteasomal and autophagic/lysosomal pathways, independently. These findings point to FoxO3 and Bnip3 as potential therapeutic targets in muscle wasting disorders and other degenerative and neoplastic diseases in which autophagy is involved.
Asunto(s)
Autofagia/fisiología , Factores de Transcripción Forkhead/metabolismo , Músculo Esquelético/citología , Músculo Esquelético/metabolismo , Animales , Autofagia/genética , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/antagonistas & inhibidores , Factores de Transcripción Forkhead/genética , Regulación de la Expresión Génica , Lisosomas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Modelos Biológicos , Atrofia Muscular/genética , Atrofia Muscular/metabolismo , Atrofia Muscular/patología , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Interferencia de ARN , Serina-Treonina Quinasas TOR , Ubiquitina/metabolismoRESUMEN
Neuromuscular synapse formation depends upon coordinated interactions between motor neurons and muscle fibers, leading to the formation of a highly specialized postsynaptic membrane and a highly differentiated nerve terminal. Synapse formation begins as motor axons approach muscles that are prepatterned in the prospective synaptic region in a manner that depends upon Lrp4, a member of the LDL receptor family, and muscle-specific kinase (MuSK), a receptor tyrosine kinase. Motor axons supply Agrin, which binds Lrp4 and stimulates further MuSK phosphorylation, stabilizing nascent synapses. How Agrin binds Lrp4 and stimulates MuSK kinase activity is poorly understood. Here, we demonstrate that Agrin binds to the N-terminal region of Lrp4, including a subset of the LDLa repeats and the first of four ß-propeller domains, which promotes association between Lrp4 and MuSK and stimulates MuSK kinase activity. In addition, we show that Agrin stimulates the formation of a functional complex between Lrp4 and MuSK on the surface of myotubes in the absence of the transmembrane and intracellular domains of Lrp4. Further, we demonstrate that the first Ig-like domain in MuSK, which shares homology with the NGF-binding region in Tropomyosin Receptor Kinase (TrKA), is required for MuSK to bind Lrp4. These findings suggest that Lrp4 is a cis-acting ligand for MuSK, whereas Agrin functions as an allosteric and paracrine regulator to promote association between Lrp4 and MuSK.
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Agrina/metabolismo , Agrina/farmacología , Inmunoglobulinas/química , Proteínas Relacionadas con Receptor de LDL/química , Proteínas Relacionadas con Receptor de LDL/metabolismo , Proteínas Tirosina Quinasas Receptoras/química , Proteínas Tirosina Quinasas Receptoras/metabolismo , Receptores Colinérgicos/química , Receptores Colinérgicos/metabolismo , Animales , Línea Celular , Activación Enzimática/efectos de los fármacos , Espacio Extracelular/metabolismo , Humanos , Modelos Moleculares , Unión Proteica/efectos de los fármacos , Estructura Terciaria de Proteína , Secuencias Repetitivas de Aminoácido , Solventes/químicaRESUMEN
Synapse formation at the neuromuscular junction (NMJ) requires an alternatively spliced variant of agrin (Z(+) agrin) that is produced only by neurons. Here, we show that Nova1 and Nova2, neuron-specific splicing factors identified as targets in autoimmune motor disease, are essential regulators of Z(+) agrin. Nova1/Nova2 double knockout mice are paralyzed and fail to cluster AChRs at the NMJ, and breeding them with transgenic mice constitutively expressing Z(+) agrin in motor neurons rescued AChR clustering. Surprisingly, however, these rescued mice remained paralyzed, while electrophysiologic studies demonstrated that the motor axon and synapse were functional-spontaneous and evoked recordings revealed synaptic transmission and muscle contraction. These results point to a proximal defect in motor neuron firing in the absence of Nova and reveal a previously unsuspected role for RNA regulation in the physiologic activation of motor neurons.