RESUMO
S-laminin/laminin beta 2, a homologue of the widely distributed laminin B1/beta 1 chain, is a major component of adult renal glomerular basement membrane (GBM). Immature GBM bears beta 1, which is replaced by beta 2 as development proceeds. In mutant mice that lack beta 2, the GBM remains rich in beta 1, suggesting that a feedback mechanism normally regulates GBM maturation. The beta 2-deficient GBM is structurally intact and contains normal complements of several collagenous and noncollagenous glycoproteins. However, mutant mice develop massive proteinuria due to failure of the glomerular filtration barrier. These results support the idea that laminin beta chains are functionally distinct although they assemble to form similar structures. Laminin beta 2-deficient mice may provide a model for human congenital or idiopathic nephrotic syndromes.
Assuntos
Glomérulos Renais/metabolismo , Laminina/deficiência , Nefrose/metabolismo , Animais , Membrana Basal/patologia , Modelos Animais de Doenças , Glomérulos Renais/patologia , Laminina/genética , Laminina/metabolismo , Camundongos , Camundongos Knockout , Nefrose/genética , Nefrose/patologiaRESUMO
Recombinant acetylcholine receptors (AChRs) expressed on the surface of cultured fibroblasts become organized into discrete membrane domains when the 43-kD postsynaptic protein (43k) is co-expressed in the same cells (Froehner, S.C., C. W. Luetje, P. B. Scotland, and J. Patrick, 1990. Neuron. 5:403-410; Phillips, W. D., M. C. Kopta, P. Blount, P. D. Gardner, J. H. Steinbach, and J. P. Merlie. 1991. Science (Wash. DC). 251:568-570). Here we show that AChRs present on the fibroblast cell surface prior to transfection of 43k are recruited into 43k-rich membrane domains. Aggregated AChRs show increased resistance to extraction with Triton X-100, suggesting a 43k-dependent linkage to the cytoskeleton. Myotubes of the mouse cell line C2 spontaneously display occasional AChR/43k-rich membrane domains that ranged in diameter up to 15 microns, but expressed many more when 43k was overexpressed following transfection of 43k cDNA. However, the membrane domains induced by recombinant 43k were predominantly small (< or = 2 microns). We were then interested in whether the cytoskeletal component, dystrophin related protein (DRP; Tinsley, J. M., D. J. Blake, A. Roche, U. Fairbrother, J. Riss, B. C. Byth, A. E. Knight, J. Kendrick-Jones, G. K. Suthers, D. R. Love, Y. H. Edwards, and K. E. Davis, 1992. Nature (Lond.). 360:591-593) contributed to the development of AChR clusters. Immunofluorescent anti-DRP staining was present at the earliest stages of AChR clustering at the neuromuscular synapse in mouse embryos and was also concentrated at the large AChR-rich domains on nontransfected C2 myotubes. Surprisingly, anti-DRP staining was concentrated mainly at the large, but not the small AChR clusters on C2 myotubes suggesting that DRP may be principally involved in permitting the growth of AChR clusters.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas de Membrana , Músculos/metabolismo , Receptores Colinérgicos/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Proteínas do Citoesqueleto/análise , Imunofluorescência , Substâncias Macromoleculares , Camundongos , Ligação Proteica , Codorniz , Receptores Colinérgicos/análise , Receptores Colinérgicos/isolamento & purificação , Proteínas Recombinantes/análise , Proteínas Recombinantes/metabolismo , Sinapses/metabolismo , Transfecção , UtrofinaRESUMO
A study has been made of the development of limb and muscle nerves in relation to the first appearance of Schwann cells in the flexor digitorum profundus (fdp) and flexor carpi ulnaris (fcu) muscles of the avian forelimb. Schwann cells were identified by immunofluorescent techniques with antibodies to the glycoprotein HNK-1. Myotubes and nerves were identified by using antibodies to myosin and to neurofilament, respectively. At stage 24/25 the brachialis longus inferior (Bli n) and superior (Bls n) nerve trunks within proximal regions of the forelimb were surrounded by Schwann cells. These cells extended in a column for a distance of approximately 100 microns beyond the growing ends of nerves. At stage 26 both interosseus nerve (in n) and the medial-ulnar nerve (m-u n) had formed from the Bli n; each of these branches was surrounded by Schwann cells, which again extended approximately 100 microns beyond the growing ends of the nerves. By stage 26/27 the fdp and fcu muscles were clearly delineated by groups of myotubes. No nerves were detected within these groups; however, Schwann cells were observed between the myotubes. At stage 27 axons had left the in n and m-u n and grown into the fdp and fcu muscles, respectively. These axons were surrounded by Schwann cells. The present observations show that Schwann cells are located ahead of the main limb and muscle nerves as they grow into the fdp and fcu muscles of the limb. It is possible that these Schwann cells play a role in guiding nerves to their correct muscles in the developing chick forelimb.
Assuntos
Neurônios Motores/fisiologia , Músculos/inervação , Crista Neural/citologia , Nervos Periféricos/embriologia , Células de Schwann/citologia , Animais , Embrião de Galinha , Imunofluorescência , Músculos/embriologia , Nervos Periféricos/citologia , Células de Schwann/imunologia , Asas de Animais/embriologiaRESUMO
A study has been made of the effects of neural crest and neural tube removal at the brachial level on the migration of Schwann cells and axons into the flexor digitorum profundus (fdp) and flexor carpi ulnaris (fcu) muscles of the avian forelimb. The identification of Schwann cells was based on the assumption that antibody HNK-1 uniquely labels these cells at the growing end of limb nerves. Myotubes and nerves were identified by using antibodies to myosin and to neurofilament protein, respectively. The removal of neural crest cells at stage 13 gave a complete Schwann cell-free embryo at the brachial level. Motor axons only grew to the base of the forelimb, forming a rudimentary plexus by stage 27, and failed to penetrate the limb. Removal of the neural tube at stage 13 did not prevent sensory axons from forming a plexus at the base of the limb; these axons subsequently developed into the brachialis longus inferior (bli n) and superior (bls n) nerves. By stage 27 the bli n had branched into the interosseus nerve (in n) and the medial-ulnar nerve (m-u n) trunks. However, unlike the result in control embryos, no nerves were detected amongst the developing fdp and fcu muscles, thus indicating that sensory axons do not grow into the muscles in the absence of motor axons. In contrast, Schwann cells were observed amongst the myotubes at the level of the in n and m-u nerve trunks. The present observations show that motor axons do not enter the limb bud and innervate limb muscles in the absence of Schwann cells. Furthermore, in the absence of motor axons (neural-tube-removed embryos) sensory axons still enter the limb (behind migrating Schwann cells) but fail to innervate developing muscles even though Schwann cells are present among the developing myotubes.
Assuntos
Axônios/fisiologia , Músculos/inervação , Sistema Nervoso/embriologia , Crista Neural/fisiologia , Células de Schwann/fisiologia , Animais , Anticorpos Monoclonais , Axônios/ultraestrutura , Embrião de Galinha , Imunofluorescência , Filamentos Intermediários/ultraestrutura , Músculos/embriologia , Subfragmentos de Miosina , Miosinas/análise , Fenômenos Fisiológicos do Sistema Nervoso , Fragmentos de Peptídeos/análise , Células de Schwann/citologia , Asas de Animais/inervaçãoRESUMO
A study has been made of the growth of cervical nerves C3-C6 to the rat diaphragm. At 11 days of embryonic age these cervical nerves first project out of the spinal cord toward the cardinal veins and later form the left and right phrenic nerve trunks. During the next 2 days, the phrenic nerves grow caudally in close association with the cardinal veins toward the diaphragm. At the growing tips of these nerve trunks the growth cones of axons were observed every 1-2 micrometers. The last axon did not project more than 2 micrometers ahead of any neighbouring axons. At 14 days the phrenic nerves reach the level of the developing diaphragm and converge into pools of premuscle cells. Previous studies have suggested that the phrenic nerve enters the premuscle masses of the diaphragm at an early developmental stage when the premuscle masses are at approximately the segmental levels C3-C6. This study shows that the phrenic nerves must grow to more caudal levels in order to reach the premuscle cells of the diaphragm. Furthermore, the leading axons of the phrenic nerve trunk do not project in a pioneering fashion, far in advance of the trailing axons.
Assuntos
Diafragma/embriologia , Nervo Frênico/embriologia , Animais , Axônios/fisiologia , Contagem de Células , Microscopia Eletrônica , Nervo Frênico/ultraestrutura , Ratos , Ratos EndogâmicosRESUMO
A study has been made of the development of muscle nerves to primary myotube clusters destined to become the flexor carpi ulnaris (fcu) and flexor digitorum profundus (fdp) muscles in the avian forelimb. Myotubes and nerves were identified by immunofluorescent techniques using antibodies to the heavy and light subunits of myosin and neurofilament, respectively. At stage 24 the main ventral nerve trunk (the brachialis longus inferior nerve; bli n) had entered the limb before the appearance of myotubes in the limb. At stage 25/26 the bli n within the ventral compartment of the forearm had given rise to the interosseus nerve (in n) and the medial-ulnar nerve (m-u n) at the junction of the stylopodium and zeugopodium. The first few myotubes of the fdp and fcu muscles were observed at this level within the ventral premuscle cell mass; however, no nerves projected toward these myotubes from either the in n or the m-u n at this time. At stage 26/27 the fcu and the fdp muscles each consisted of clusters of 20-40 myotubes; each cluster was clearly delineated within the ventral premuscle cell mass. By this time small groups of axons had left the in n and the m-u n to grow into the fdp and fcu myotube clusters, respectively; these axons formed the muscle nerves. At stage 28/29 the number of primary myotubes in the clusters composing the fdp and fcu muscles had greatly increased, as did the size of the muscle nerves; each muscle was still clearly identifiable within the ventral muscle mass. By stage 32 the fdp and fcu muscles had clearly separated and the muscle nerves had divided into several well-spaced branches within each muscle. The present observations show that the main nerve trunks grow into the limb before the formation of myotubes expressing myosin isozymes. When myotubes do form they appear in small clusters at specific sites within the premuscle mass, before muscle nerves appear; a distinct muscle is destined to form from each of these clusters. Muscle nerves first branch from the main limb nerves when the myotube cluster contains more than about ten myotubes.
Assuntos
Neurônios Motores/fisiologia , Músculos/inervação , Asas de Animais/embriologia , Animais , Anticorpos Monoclonais , Diferenciação Celular , Embrião de Galinha , Desenvolvimento Embrionário e Fetal , Imunofluorescência , Proteínas de Filamentos Intermediários/análise , Camundongos , Neurônios Motores/análise , Músculos/análise , Músculos/embriologia , Miosinas/análise , Proteínas de Neurofilamentos , Coloração e Rotulagem , Asas de Animais/inervaçãoRESUMO
Inhibition of programmed cell death of motoneurons during embryonic development requires the presence of their target muscle and coincides with the initial stages of synaptogenesis. To evaluate the role of synapse formation on motoneuron survival during embryonic development, we counted the number of motoneurons in rapsyn-deficient mice. Rapsyn is a 43 kDa protein needed for the formation of postsynaptic specialisations at vertebrate neuromuscular synapses. Here we show that the rapsyn-deficient mice have a significant increase in the number of motoneurons in the brachial lateral motor column during the period of naturally occurring programmed cell death compared to their wild-type littermates. In addition, we observed an increase in intramuscular axonal branching in the rapsyn-deficient diaphragms compared to their wild-type littermates at embryonic day 18.5. These results suggest that deficits in the formation of the postsynaptic specialisation at the neuromuscular synapse, brought about by the absence of rapsyn, are sufficient to induce increases in both axonal branching and the survival of the innervating motoneuron. Moreover, these results support the idea that skeletal muscle activity through effective synaptic transmission and intramuscular axonal branching are major mechanisms that regulate motoneuron survival during development.
Assuntos
Diferenciação Celular/genética , Sobrevivência Celular/genética , Neurônios Motores/metabolismo , Proteínas Musculares/deficiência , Junção Neuromuscular/embriologia , Medula Espinal/embriologia , Membranas Sinápticas/metabolismo , Animais , Apoptose/genética , Axônios/metabolismo , Axônios/ultraestrutura , Contagem de Células/estatística & dados numéricos , Tamanho Celular/genética , Diafragma/citologia , Diafragma/inervação , Diafragma/metabolismo , Feminino , Camundongos , Camundongos Knockout , Neurônios Motores/citologia , Proteínas Musculares/genética , Junção Neuromuscular/citologia , Junção Neuromuscular/metabolismo , Receptores Colinérgicos/genética , Receptores Colinérgicos/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Membranas Sinápticas/ultraestruturaRESUMO
The expression and subcellular localisation of dynamin and syntaxin were examined during the periods of motor neuron development and neuromuscular synaptogenesis in the mouse embryo. Both dynamin and syntaxin could be detected by immunoblotting in the spinal cord at embryonic day 10 (E10; 2 days before axon outgrowth) and at all subsequent ages examined. Reverse transcription and polymerase chain reaction (RT-PCR) identified low levels of all three carboxy-terminal splicing forms of dynamin I in spinal cord from as early as E10. During the period of maturation of spinal neurons, from E10 to the first postnatal day (P0), the short carboxy-terminal splicing form of dynamin I (dynamin I*b) was up-regulated, as was dynamin III, relative to dynamin II mRNA. Syntaxin immunostaining became colocalized with the synaptic vesicle protein, SV2, at neuromuscular synapses within 12 hours of the commencement of synapse formation and throughout subsequent development. In contrast, dynamin, which is important for activity-dependent synaptic vesicle recycling and, thus, sustained neurotransmission, could not be detected at most newly formed synapses until several days after synapse formation. The delayed appearance of dynamin at the synapse, thus, heralds the neonatal development of robust synaptic transmission at the neuromuscular junction.
Assuntos
Animais Recém-Nascidos/metabolismo , Embrião de Mamíferos/fisiologia , GTP Fosfo-Hidrolases/metabolismo , Proteínas de Membrana/metabolismo , Sistema Nervoso/embriologia , Junção Neuromuscular/embriologia , Sinapses/fisiologia , Animais , Dinamina I , Dinamina III , Dinaminas , Embrião de Mamíferos/metabolismo , Desenvolvimento Embrionário e Fetal/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Sistema Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Isoformas de Proteínas/metabolismo , Proteínas Qa-SNARE , Medula Espinal/embriologia , Distribuição Tecidual/fisiologiaRESUMO
Recent studies demonstrate that P2X7 receptor subunits (P2X7RS) are present at central and peripheral synapses and suggest that P2X7RS can regulate transmitter release. In brainstem slices from 15 to 26 day old pentobarbitone-anesthetized mice, we examined the effect of P2X7RS activation on excitatory postsynaptic currents (EPSCs) recorded from hypoglossal motoneurons using whole-cell patch clamp techniques. After blockade of most P2X receptors with suramin (which is inactive at P2X7RS) and of adenosine receptors with 8-phenyltheophylline (8PT), bath application of the P2X receptor agonist 3'-0-(4-benzoyl)ATP (BzATP) elicited a 40.5+/-16.0% (mean+/-S.E.M., n = 8, P = 0.039) increase in evoked EPSC amplitude and significantly reduced paired pulse facilitation of evoked EPSCs. This response to BzATP (with suramin and 8PT present) was completely blocked by prior application of Brilliant Blue G (200 nM or 2 microM), a P2X7RS antagonist. In contrast, BzATP application with suramin and 8PT present did not alter miniature EPSC frequency or amplitude when action potentials were blocked with tetrodotoxin. These electrophysiological results suggest that P2X7RS activation increases central excitatory transmitter release via presynaptic mechanisms, confirming previous indirect measures of enhanced transmitter release. We suggest that possible presynaptic mechanisms underlying enhancement of evoked transmitter release by P2X7RS activation are modulation of action potential width or an increase in presynaptic terminal excitability, due to subthreshold membrane depolarization which increases the number of terminals releasing transmitter in response to stimulation.
Assuntos
Trifosfato de Adenosina/análogos & derivados , Tronco Encefálico/fisiologia , Nervo Hipoglosso/fisiologia , Neurônios Motores/fisiologia , Terminações Pré-Sinápticas/fisiologia , Receptores Purinérgicos P2/fisiologia , Transmissão Sináptica/fisiologia , Trifosfato de Adenosina/farmacologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Feminino , Masculino , Camundongos , Camundongos Endogâmicos , Isoformas de Proteínas/fisiologia , Receptores Purinérgicos P2X7 , Transmissão Sináptica/efeitos dos fármacosRESUMO
Early endosomal antigen 1 (EEA1) is known to be a marker of early endosomes and in cultured hippocampal neurons it preferentially localizes to the dendritic but not the axonal compartment. We show in cultured dorsal root ganglia and superior cervical ganglia neurons that EEA1 localizes to the cell bodies and the neurites of both sensory and sympathetic neurons. We then show in vivo using a ligated rat sciatic nerve that EEA1 significantly accumulates on the proximal side and not on the distal side of the ligation. This suggests that EEA1 is transported in the anterograde direction in axons either as part of the homeostatic process or to the nerve ligation site in response to nerve injury.
Assuntos
Transporte Axonal/fisiologia , Proteínas de Membrana/metabolismo , Neurônios Aferentes/metabolismo , Nervo Isquiático/citologia , Nervo Isquiático/metabolismo , Animais , Animais Recém-Nascidos , Axônios/química , Axônios/metabolismo , Endossomos/metabolismo , Imunofluorescência , Gânglios Espinais/citologia , Homeostase/fisiologia , Ligadura , Masculino , Proteínas de Membrana/análise , Camundongos , Camundongos Endogâmicos BALB C , Neurônios Aferentes/química , Neurônios Aferentes/ultraestrutura , Ratos , Ratos Wistar , Nervo Isquiático/lesões , Gânglio Cervical Superior/citologia , Proteínas de Transporte VesicularRESUMO
Formation of the skeletal neuromuscular junction is a multi-step process that requires communication between the nerve and muscle. Studies in many laboratories have led to identification of factors that seem likely to mediate these interactions. 'Knock-out' mice have now been generated with mutations in several genes that encode candidate transsynaptic messengers and components of their effector mechanisms. Using these mice, it is possible to test hypotheses about the control of synaptogenesis. Here, we review our studies on neuromuscular development in mutant mice lacking agrin alpha CGRP, rapsyn, MuSK, dystrophin, dystrobrevin, utrophin, laminin alpha 5, laminin beta 2, collagen alpha 3 (IV), the acetylcholine receptor epsilon subunit, the collagenous tail of acetylcholinesterase, fibroblast growth factor-5, the neural cell adhesion molecule, and tenascin-C.
Assuntos
Engenharia Genética , Proteínas Musculares/genética , Proteínas do Tecido Nervoso/genética , Junção Neuromuscular/fisiologia , Agrina/genética , Animais , Camundongos , Camundongos Knockout , Mutação , Isoformas de Proteínas/genéticaRESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease, characterized by the progressive loss of motor neurons within the central nervous system. Neural degeneration and inflammatory processes, including activation of the complement system are hallmarks of this pathology. Our past work in ALS animal models (hSOD1 G93A rodents) has revealed that blockade of the receptor for complement activation fragment C5a (C5aR), improves ALS-like symptoms and extends survival. We now show that the levels of C5a and C5b-9, but not C3a nor C4a, are significantly elevated in plasma from ALS patients compared to healthy controls. C5a was also elevated within leukocytes from ALS patients suggesting heightened C5a receptor interaction. Overall, these findings indicate that there is enhanced peripheral immune complement terminal pathway activation in ALS, which may have relevance in the disease process.
Assuntos
Esclerose Lateral Amiotrófica/sangue , Complemento C5a/metabolismo , Complexo de Ataque à Membrana do Sistema Complemento/metabolismo , Adulto , Idoso , Esclerose Lateral Amiotrófica/patologia , Anafilatoxinas/metabolismo , Ensaio de Imunoadsorção Enzimática , Feminino , Regulação da Expressão Gênica , Humanos , Leucócitos/metabolismo , Masculino , Pessoa de Meia-Idade , Estatísticas não ParamétricasRESUMO
Rapsyn is a protein on the cytoplasmic face of the postsynaptic membrane of skeletal muscle that is essential for clustering acetylcholine receptors (AChR). Here we show that transfection of rapsyn cDNA can restore AChR clustering function to muscle cells cultured from rapsyn deficient (KORAP) mice. KORAP myotubes displayed no AChR aggregates before or after treatment with neural agrin. After transfection with rapsyn expression plasmid, some KORAP myotubes expressed rapsyn at physiological levels. These formed large AChR-rapsyn clusters in response to agrin, just like wild-type myotubes. KORAP myotubes that overexpressed rapsyn formed only scattered AChR-rapsyn microaggregates, irrespective of agrin treatment. KORAP cells were then transfected with mutant forms of rapsyn. A deletion mutant lacking residues 16-254 formed rapsyn microaggregates, but failed to aggregate AChRs. Substitution mutation to the C-terminal serine phosphorylation site of rapsyn (M43(D405,D406)) did not impair the response to agrin, showing that differential phosphorylation of this site is unlikely to mediate agrin-induced clustering. The results indicate that rapsyn expression is essential for agrin-induced AChR clustering but that its overexpression inhibits this pathway. The approach of using rapsyn-deficient muscle cells opens the way for defining the role of rapsyn in agrin-induced AChR clustering.
Assuntos
Agrina/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas Musculares/genética , Receptores Colinérgicos/metabolismo , Animais , Linhagem Celular Transformada , Proteínas do Citoesqueleto/análise , Proteínas do Citoesqueleto/metabolismo , Feminino , Deleção de Genes , Expressão Gênica/fisiologia , Masculino , Proteínas de Membrana/análise , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Mutantes , Fibras Musculares Esqueléticas/química , Proteínas Musculares/análise , Proteínas Musculares/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/inervação , Mutagênese/fisiologia , Fragmentos de Peptídeos/genética , Fosforilação , Serina/metabolismo , Transfecção , UtrofinaRESUMO
Synapse formation requires a complex interchange of information between the pre- and postsynaptic partners. At the skeletal neuromuscular junction, some of this information is contained in the basal lamina (BL), which runs through the synaptic cleft between the motor nerve terminal and the muscle fibre. During regeneration following injury, components of synaptic BL can trigger several features of postsynaptic differentiation in the absence of the nerve terminal, and of presynaptic differentiation in the absence of the muscle fibre. One nerve-derived component of synaptic BL, agrin, is known to affect postsynaptic differentiation, but no muscle-derived components have yet been shown to influence motor nerve terminals. A candidate for such a role is s-laminin (also called laminin beta 2), a homologue of the B1 (beta 1) chain of the widely distributed BL glycoprotein, laminin. s-Laminin is synthesized by muscle cells and concentrated in synaptic BL. In vitro, recombinant s-laminin fragments are selectively adhesive for motor neuron-like cells, inhibit neurite outgrowth promoted by other matrix molecules, and act as a 'stop signal' for growing neurites. By generating and characterizing mice with a targeted mutation of the s-laminin gene, we show here that s-laminin regulates formation of motor nerve terminals.
Assuntos
Laminina/fisiologia , Junção Neuromuscular/embriologia , Potenciais de Ação , Animais , Diferenciação Celular/genética , Laminina/deficiência , Laminina/genética , Camundongos , Neurônios Motores/fisiologia , Neurônios Motores/ultraestrutura , Mutação , Junção Neuromuscular/ultraestruturaRESUMO
Rapsyn is a 43-kDa cytoplasmic protein that clusters nicotinic acetylcholine receptors (AChR) in the postsynaptic membrane. Here we examine the effect of rapsynmediated AChR clustering on the metabolic stability of the AChR. When transfected into QT-6 fibroblasts, cell surface AChRs (alpha, beta, epsilon, and delta subunit combination) pulse labeled with 125I-alpha-bungarotoxin were degraded with a half-life of 16.4 +/- 1.1 h (mean +/- SEM). Cotransfection of rapsyn with AChR caused extensive AChR clustering and increased AChR half-life to 20.5 +/- 1.0 h. Anti-AChR antibodies such as mab 35 cause an increased AChR degradation often associated with myasthenia gravis: 80.8 +/- 2.5% of AChRs labeled at zero time were degraded over a 12-h period. Contransfection of rapsyn reduced this AChR loss to 66.4 +/- 3.8%. Rapsyn also reduced normal AChR degradation, from 53.2 +/- 2.1 to 44.2 +/- 2.2%. Muscle cell lines from wild-type myotubes displayed few AChR clusters, but treatment with neural agrin increased the number of AChR clusters 30-fold. Clustering was accompanied by reductions in AChR degradation (both in the presence and absence of mab 35) similar in magnitude to those produced by overexpression of rapsyn in QT-6 cells. In rapsyn-deficient myotubes, treatment with neural agrin neither caused AChR clustering nor reduced AChR degradation. Thus neural agrin may slow AChR degradation by inducing the rapsyn-dependent clustering of AChRs.
Assuntos
Agrina/farmacologia , Proteínas Musculares/farmacologia , Receptores Colinérgicos/efeitos dos fármacos , Receptores Colinérgicos/metabolismo , Animais , Anticorpos/imunologia , Células CHO , Linhagem Celular , Cricetinae , Fibroblastos/metabolismo , Imunofluorescência , Proteínas Musculares/genética , Músculos/citologia , Músculos/metabolismo , Mutação , Agregação de Receptores/efeitos dos fármacos , Receptores Colinérgicos/imunologia , Receptores Nicotínicos/genéticaRESUMO
Developing motor axons induce synaptic specializations in muscle fibers, including preferential transcription of acetylcholine receptor (AChR) subunit genes by subsynaptic nuclei. One candidate nerve-derived signaling molecule is AChR-inducing activity (ARIA)/heregulin, a ligand of the erbB family of receptor tyrosine kinases. Here, we asked whether ARIA and erbB kinases are expressed in patterns compatible with their proposed signaling roles. In developing muscle, ARIA was present not only at synaptic sites, but also in extrasynaptic regions of the muscle fiber. ARIA was synthesized, rather than merely taken up, by muscle cells, as indicated by the presence of ARIA mRNA in muscle and of ARIA protein in a clonal muscle cell line. ARIA-responsive myotubes expressed both erbB2 and erbB3, but little EGFR/erbB1 or erbB4. In adults, erbB2 and erbB3 were localized to the postsynaptic membrane. ErbB3 was restricted to the postsynaptic membrane perinatally, at a time when ARIA was still broadly distributed. Thus, our data are consistent with a model in which ARIA interacts with erbB kinases on the muscle cell surface to provide a local signal that induces synaptic expression of AChR genes. However, much of the ARIA is produced by muscle, not nerve, and the spatially restricted response may result from the localization of erbB kinases as well as of ARIA. Finally, we show that erbB3 is not concentrated at synaptic sites in mutant mice that lack rapsyn, a cytoskeletal protein required for AChR clustering, suggesting that pathways for synaptic AChR expression and clustering interact.
Assuntos
Envelhecimento/fisiologia , Receptores ErbB/biossíntese , Expressão Gênica , Músculo Esquelético/metabolismo , Proteínas do Tecido Nervoso/biossíntese , Proteínas Proto-Oncogênicas/biossíntese , Receptor ErbB-2/biossíntese , Receptor ErbB-3/biossíntese , Receptores Colinérgicos/biossíntese , Sinapses/fisiologia , Sequência de Aminoácidos , Animais , Axônios/fisiologia , Sequência de Bases , Primers do DNA , Feminino , Humanos , Substâncias Macromoleculares , Masculino , Mamíferos , Camundongos , Dados de Sequência Molecular , Denervação Muscular , Desenvolvimento Muscular , Fibras Musculares Esqueléticas/metabolismo , Músculo Esquelético/crescimento & desenvolvimento , Neuregulina-1 , Gravidez , Ratos , Ratos Wistar , Homologia de Sequência de AminoácidosRESUMO
Rapsyn is a key molecule involved in the formation of postsynaptic specializations at the neuromuscular junction, in its absence there are both pre- and post-synaptic deficits including failure to cluster acetylcholine receptors. Recently we have documented increases in both nerve-muscle branching and numbers of motoneurons, suggesting alterations in skeletal muscle derived trophic support for motoneurons. The aim of the present study was to evaluate the contribution of target derived trophic factors to increases in motoneuron branching and number, in rapsyn deficient mice that had their postsynaptic specializations disrupted. We have used reverse transcription-polymerase chain reaction and Western blot to document the expression of known trophic factors and their receptors in muscle, during the period of synapse formation in rapsyn deficient mouse embryos. We found that the mRNA levels for ciliary neurotrophic factor (CNTF) was decreased in the rapsyn deficient muscles compared with litter mate controls although those for NGF, BDNF, NT-3 and TGF-beta2 did not differ. We found that both the mRNA and the protein expression for suppressor of cytokine signaling 3 (SOCS3) decreased although janus kinase 2 (JAK2) did not change in the rapsyn deficient muscles compared with litter mate controls. These results suggest that failure to form postsynaptic specializations in rapsyn deficient mice has altered the CNTF cytokine signaling pathway within skeletal muscle, the target for motoneurons. This alteration may in part, account for the increased muscle nerve branching and motoneuron survival seen in rapsyn deficient mice.
Assuntos
Fator Neurotrófico Ciliar/fisiologia , Proteínas Musculares/genética , Proteínas Proto-Oncogênicas , Proteínas Repressoras , Transdução de Sinais/fisiologia , Fatores de Transcrição , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Primers do DNA , Diafragma/inervação , Diafragma/fisiologia , Expressão Gênica/fisiologia , Janus Quinase 2 , Camundongos , Camundongos Knockout , Neurônios Motores/fisiologia , Músculo Esquelético/inervação , Músculo Esquelético/fisiologia , Fator de Crescimento Neural/genética , Neurotrofina 3/genética , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/isolamento & purificação , Proteínas/genética , Proteínas/isolamento & purificação , RNA Mensageiro/análise , Proteína 3 Supressora da Sinalização de Citocinas , Proteínas Supressoras da Sinalização de CitocinaRESUMO
Interaction of alpha 4 integrins with vascular cell adhesion molecule-1 (VCAM-1) is classically important for immune function. However, we found recently that these receptors have a second role, in embryogenesis, where they mediate cell-cell interactions that are important for skeletal muscle differentiation. Here, we present evidence of an expanding role for these receptors in murine development. alpha 4 and VCAM-1 were found at embryonic sites of hematopoiesis, suggesting a role for these receptors during embryogenesis that parallels their hematopoietic function in adult bone marrow. During angiogenesis in the lung, alpha 4 and VCAM-1 were found on mesenchyme that gives rise to vascular endothelium and smooth muscle. alpha 4 persisted on the smooth muscle and the endothelium of newly forming vessels where it colocalized with its extracellular matrix ligand, fibronectin (FN). These patterns suggest several roles for alpha 4 integrins and their ligands in angiogenesis. alpha 4 was also found on neural crest derivatives where it colocalized with FN. alpha 4 was expressed selectively on cells in the dorsal root ganglia: it was apparent along ventral projections, but absent from dorsal projections, suggesting that alpha 4 integrins could be involved in defining neuronal fates. Although VCAM-1 was not expressed on most neural crest derivatives, it was found in the neural crest-derived outflow tract of the embryonic heart, where it colocalized with alpha 4. These results imply that alpha 4 integrins and their ligands could be important for migration or differentiation of neural crest. alpha 4 was also expressed on embryonic retina and FN was found on inductive mesenchyme surrounding the eye, suggesting a role for these proteins in eye development. Finally, based on their patterns of expression, we conclude that VCAM-1 only participates in a subset of interactions involving alpha 4 integrins, whereas FN appears to be the more general ligand.
Assuntos
Envelhecimento/fisiologia , Moléculas de Adesão Celular/fisiologia , Desenvolvimento Embrionário e Fetal , Endotélio Vascular/metabolismo , Fibronectinas/fisiologia , Integrinas/fisiologia , Músculo Liso Vascular/metabolismo , Animais , Moléculas de Adesão Celular/análise , Moléculas de Adesão Celular/biossíntese , Endotélio Vascular/embriologia , Endotélio Vascular/crescimento & desenvolvimento , Feminino , Fibronectinas/análise , Fibronectinas/biossíntese , Expressão Gênica , Coração/embriologia , Coração/crescimento & desenvolvimento , Integrina alfa4 , Integrinas/análise , Integrinas/biossíntese , Pulmão/embriologia , Pulmão/crescimento & desenvolvimento , Pulmão/metabolismo , Camundongos , Desenvolvimento Muscular , Músculo Liso Vascular/embriologia , Músculo Liso Vascular/crescimento & desenvolvimento , Miocárdio/metabolismo , Especificidade de Órgãos , Gravidez , Molécula 1 de Adesão de Célula Vascular , Saco Vitelino/metabolismoRESUMO
Of numerous synaptic components that have been identified, perhaps the best-studied are the nicotinic acetylcholine receptors (AChRs) of the vertebrate neuromuscular junction. AChRs are diffusely distributed on embryonic myotubes, but become highly concentrated (approximately 10,000 microns-2) in the postsynaptic membrane as development proceeds. At least two distinct processes contribute to this accumulation. One is local synthesis: subsynaptic muscle nuclei transcribe AChR subunit genes at higher rates than extra-synaptic nuclei, so AChR messenger RNA is concentrated near synaptic sites. Second, once AChRs have been inserted in the membrane, they form high-density clusters by tethering to a subsynaptic cytoskeletal complex. A key component of this complex is rapsyn, a peripheral membrane protein of relative molecular mass 43K (refs 4, 5), which is precisely colocalized with AChRs at synaptic sites from the earliest stages of neuromuscular synaptogenesis. In heterologous systems, expression of recombinant rapsyn leads to clustering of diffusely distributed AChRs, suggesting that rapsyn may control formation of clusters. To assess the role of rapsyn in vivo, we generated and characterized mutant mice with a targeted disruption of the Rapsyn gene. We report that rapsyn is essential for the formation of AChR clusters, but that synapse-specific transcription of AChR subunit genes can proceed in its absence.
Assuntos
Proteínas Musculares/fisiologia , Junção Neuromuscular/embriologia , Receptores Nicotínicos/fisiologia , Membranas Sinápticas/fisiologia , Animais , Axônios/fisiologia , Membrana Basal/fisiologia , Linhagem Celular , Camundongos , Camundongos Transgênicos , Proteínas Musculares/deficiência , Proteínas Musculares/genética , Mutagênese , Junção Neuromuscular/ultraestrutura , Receptores Nicotínicos/deficiência , Receptores Nicotínicos/genética , Membranas Sinápticas/ultraestrutura , Transcrição GênicaRESUMO
During neuromuscular synapse formation, motor axons induce clustering of acetylcholine receptors (AChRs) in the muscle fiber membrane. The protein agrin, originally isolated from the basal lamina of the synaptic cleft, is synthesized and secreted by motoneurons and triggers formation of AChR clusters on cultured myotubes. We show here postsynaptic AChR aggregates are markedly reduced in number, size, and density in muscles of agrin-deficient mutant mice. These results support the hypothesis that agrin is a critical organizer of postsynaptic differentiation does occur in the mutant, suggesting the existence of a second-nerve-derived synaptic organizing signal. In addition, we show that intramuscular nerve branching and presynaptic differentiation are abnormal in the mutant, phenotypes which may reflect either a distinct effect of agrin or impaired retrograde signaling from a defective postsynaptic apparatus.