RESUMO
Muscle denervation at the neuromuscular junction (NMJ), the essential synapse between motor neuron and skeletal muscle, is associated with age-related motor impairment. Therefore, improving muscle innervation at aged NMJs may be an effective therapeutic strategy for treating the impairment. We previously demonstrated that the muscle protein Dok-7 plays an essential role in NMJ formation, and, indeed, its forced expression in muscle enlarges NMJs. Moreover, therapeutic administration of an adeno-associated virus vector encoding human Dok-7 (DOK7 gene therapy) suppressed muscle denervation and enhanced motor activity in a mouse model of amyotrophic lateral sclerosis (ALS). Here, we show that DOK7 gene therapy significantly enhances motor function and muscle strength together with NMJ innervation in aged mice. Furthermore, the treated mice showed greatly increased compound muscle action potential (CMAP) amplitudes compared with the controls, suggesting enhanced neuromuscular transmission. Thus, therapies aimed at enhancing NMJ innervation have potential for treating age-related motor impairment.
RESUMO
Congenital myasthenic syndromes (CMS) are characterized by fatigable muscle weakness resulting from impaired neuromuscular transmission. ß2-adrenergic agonists are an effective treatment for DOK7-CMS. DOK7 is a component within the AGRN-LRP4-MUSK-DOK7 signalling pathway that is key for the formation and maintenance of the synaptic structure of the neuromuscular junction (NMJ). The precise mechanism of action of ß2-adrenergic agonists at the NMJ is not fully understood. In this study, we investigated whether ß2-adrenergic agonists improve both neurotransmission and structural integrity of the NMJ in a mouse model of DOK7-CMS. Ex-vivo electrophysiological techniques and microscopy of the NMJ were used to study the effect of salbutamol, a ß2-adrenergic agonist, on synaptic structure and function. DOK7-CMS model mice displayed a severe phenotype with reduced weight gain and perinatal lethality. Salbutamol treatment improved weight gain and survival in DOK7 myasthenic mice. Model animals had fewer active NMJs, detectable by endplate recordings, compared with age-matched wild-type littermates. Salbutamol treatment increased the number of detectable NMJs during endplate recording. Correspondingly, model mice had fewer acetylcholine receptor-stained NMJs detected by fluorescent labelling, but following salbutamol treatment an increased number were detectable. The data demonstrate that salbutamol can prolong survival and increase NMJ number in a severe model of DOK7-CMS.
Assuntos
Albuterol/farmacologia , Proteínas Musculares/genética , Síndromes Miastênicas Congênitas/tratamento farmacológico , Junção Neuromuscular/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Feminino , Humanos , Camundongos , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/patologia , Junção Neuromuscular/metabolismo , Gravidez , Receptores Colinérgicos/genética , Transdução de Sinais/efeitos dos fármacos , Transmissão Sináptica/efeitos dos fármacosRESUMO
The neuromuscular junction(NMJ)is the synapse between a motor neuron and the skeletal muscle that is essential for muscle contraction. Impairments at the NMJ lead to neuromuscular-transmission pathologies characterized by fatigable muscle weakness. Muscle-specific receptor tyrosine kinase MuSK plays key roles in NMJ formation. Over the past decade, studies examining the NMJ formation signals have identified molecules involved in the signaling pathways and have promoted a better understanding of characteristic molecular mechanisms for MuSK activation. Unlike many other receptor tyrosine kinases, MuSK is regulated by the cytoplasmic activator Dok-7 in addition to the extracellular activator agrin. It is well established that all of these molecules are indispensable in the formation and maintenance of the NMJ. In this chapter, we review molecular signaling, particularly MuSK signaling, in the formation of the NMJ and the altered molecular signaling associated with neuromuscular disorders.
Assuntos
Homeostase , Doenças Musculoesqueléticas/metabolismo , Junção Neuromuscular/metabolismo , Transdução de Sinais , Animais , HumanosRESUMO
Bone mass is determined by coordinated acts of osteoblasts and osteoclasts, which control bone formation and resorption, respectively. Osteoclasts are multinucleated, macrophage/monocyte lineage cells from bone marrow. The Dok-family adaptors Dok-1, Dok-2 and Dok-3 are expressed in the macrophage/monocyte lineage and negatively regulate many signaling pathways, implying roles in osteoclastogenesis. Indeed, mice lacking Dok-1 and Dok-2, the closest homologues with redundant functions, develop osteopenia with increased osteoclast counts compared to the wild-type controls. Here, we demonstrate that Dok-3 knockout (KO) mice also develop osteopenia. However, Dok-3 KO, but not Dok-1/-2 double-KO (DKO), mice develop larger osteoclasts within the normal cell-count range, suggesting a distinctive role for Dok-3. Indeed, Dok-3 KO, but not Dok-1/-2 DKO, bone marrow-derived cells (BMDCs) generated larger osteoclasts with more nuclei due to augmented cell-to-cell fusion in vitro. In addition, while Dok-1/-2 DKO BMDCs generated more osteoclasts, Dok-1/-2/-3 triple-KO (TKO) BMDCs generated osteoclasts increased in both number and size. Furthermore, Dok-1/-2/-3 TKO mice showed the combined effects of Dok-3 and Dok-1/-2 deficiency: severe osteopenia with more and larger osteoclasts. Together, our findings demonstrate that Dok-3 and Dok-1/-2 play distinctive but cooperative roles in osteoclastogenesis and protect mice from osteopenia, providing physiological and pathophysiological insight into bone homeostasis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Doenças Ósseas Metabólicas/prevenção & controle , Proteínas de Ligação a DNA/fisiologia , Osteoclastos/citologia , Osteogênese , Fosfoproteínas/fisiologia , Proteínas de Ligação a RNA/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Células da Medula Óssea/citologia , Contagem de Células , Técnicas de Cultura de Células , Fusão Celular , Proliferação de Células , Tamanho Celular , Proteínas de Ligação a DNA/genética , Camundongos , Camundongos Knockout , Fosfoproteínas/genética , Proteínas de Ligação a RNA/genéticaRESUMO
As the synapse between a motor neuron and skeletal muscle, the neuromuscular junction (NMJ) is required for muscle contraction. The formation and maintenance of NMJs are controlled by the muscle-specific receptor kinase MuSK. Dok-7 is the essential cytoplasmic activator of MuSK, and indeed mice lacking Dok-7 form no NMJs. Moreover, DOK7 gene mutations underlie DOK7 myasthenia, an NMJ synaptopathy. Previously, we failed to detect MuSK activation in myotubes by Dok-7 mutated in the N-terminal pleckstrin homology (PH) or phosphotyrosine binding (PTB) domain or that lacked the C-terminal region (Dok-7-ΔC). Here, we found by quantitative analysis that Dok-7-ΔC marginally, but significantly, activated MuSK in myotubes, unlike the PH- or PTB-mutant. Purified, recombinant Dok-7-ΔC, but not other mutants, also showed marginal ability to activate MuSK's cytoplasmic portion, carrying the kinase domain. Consistently, forced expression of Dok-7-ΔC rescued Dok-7-deficient mice from neonatal lethality caused by the lack of NMJs, indicating restored MuSK activation and NMJ formation. However, these mice showed only marginal activation of MuSK and died by 3 weeks of age apparently due to an abnormally small number and size of NMJs. Thus, Dok-7's C-terminal region plays a key, but not fully essential, role in MuSK activation and NMJ formation.
Assuntos
Proteínas Musculares/química , Proteínas Musculares/metabolismo , Junção Neuromuscular/enzimologia , Junção Neuromuscular/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Células Cultivadas , CamundongosRESUMO
The motoneural control of skeletal muscle contraction requires the neuromuscular junction (NMJ), a midmuscle synapse between the motor nerve and myotube. The formation and maintenance of NMJs are orchestrated by the muscle-specific receptor tyrosine kinase (MuSK). Motor neuron-derived agrin activates MuSK via binding to MuSK's coreceptor Lrp4, and genetic defects in agrin underlie a congenital myasthenic syndrome (an NMJ disorder). However, MuSK-dependent postsynaptic differentiation of NMJs occurs in the absence of a motor neuron, indicating a need for nerve/agrin-independent MuSK activation. We previously identified the muscle protein Dok-7 as an essential activator of MuSK. Although NMJ formation requires agrin under physiological conditions, it is dispensable for NMJ formation experimentally in the absence of the neurotransmitter acetylcholine, which inhibits postsynaptic specialization. Thus, it was hypothesized that MuSK needs agrin together with Lrp4 and Dok-7 to achieve sufficient activation to surmount inhibition by acetylcholine. Here, we show that forced expression of Dok-7 in muscle enhanced MuSK activation in mice lacking agrin or Lrp4 and restored midmuscle NMJ formation in agrin-deficient mice, but not in Lrp4-deficient mice, probably due to the loss of Lrp4-dependent presynaptic differentiation. However, these NMJs in agrin-deficient mice rapidly disappeared after birth, and postsynaptic specializations emerged ectopically throughout myotubes whereas exogenous Dok-7-mediated MuSK activation was maintained. These findings demonstrate that the MuSK activator agrin plays another role essential for the postnatal maintenance, but not for embryonic formation, of NMJs and also for the postnatal, but not prenatal, midmuscle localization of postsynaptic specializations, providing physiological and pathophysiological insight into NMJ homeostasis.
Assuntos
Agrina/fisiologia , Junção Neuromuscular/enzimologia , Receptores Proteína Tirosina Quinases/metabolismo , Agrina/deficiência , Agrina/genética , Processamento Alternativo , Animais , Diafragma/embriologia , Diafragma/crescimento & desenvolvimento , Ativação Enzimática , Feminino , Proteínas Relacionadas a Receptor de LDL , Longevidade/genética , Masculino , Camundongos , Camundongos Transgênicos , Fibras Musculares Esqueléticas/enzimologia , Fibras Musculares Esqueléticas/fisiologia , Proteínas Musculares/deficiência , Proteínas Musculares/fisiologia , Junção Neuromuscular/embriologia , Junção Neuromuscular/crescimento & desenvolvimento , Doenças da Junção Neuromuscular/enzimologia , Doenças da Junção Neuromuscular/genética , Doenças da Junção Neuromuscular/fisiopatologia , Fosforilação , Densidade Pós-Sináptica/fisiologia , Processamento de Proteína Pós-Traducional , Receptores Colinérgicos/fisiologia , Receptores de LDL/deficiência , Receptores de LDL/fisiologia , Proteínas Recombinantes de Fusão/metabolismo , Teste de Desempenho do Rota-RodRESUMO
Aft1p is an iron-responsive transcriptional activator that plays a central role in the regulation of iron metabolism in Saccharomyces cerevisiae. Aft1p is regulated by accelerated nuclear export in the presence of iron, mediated by Msn5p. However, the transcriptional activity of Aft1p is suppressed under iron-replete conditions in the Δmsn5 strain, although Aft1p remains in the nucleus. Aft1p dissociates from its target promoters under iron-replete conditions due to an interaction between Aft1p and the monothiol glutaredoxin Grx3p or Grx4p (Grx3/4p). The binding of Grx3/4p to Aft1p is induced by iron repletion and requires binding of an iron-sulfur cluster to Grx3/4p. The mitochondrial transporter Atm1p, which has been implicated in the export of iron-sulfur clusters and related molecules, is required not only for iron binding to Grx3p but also for dissociation of Aft1p from its target promoters. These results suggest that iron binding to Grx3p (and presumably Grx4p) is a prerequisite for the suppression of Aft1p. Since Atm1p plays crucial roles in the delivery of iron-sulfur clusters from the mitochondria to the cytoplasm and nucleus, these results support the previous observations that the mitochondrial iron-sulfur cluster assembly machinery is involved in cellular iron sensing.
Assuntos
Ferro/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Sítios de Ligação/genética , Expressão Gênica , Genes Fúngicos , Glutarredoxinas/genética , Glutarredoxinas/metabolismo , Proteínas Ferro-Enxofre/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Modelos Biológicos , Mutação , Oxirredutases/genética , Oxirredutases/metabolismo , Regiões Promotoras Genéticas , Transporte Proteico , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Fatores de Transcrição/genéticaAssuntos
Células/metabolismo , Ferro/metabolismo , Mitocôndrias/fisiologia , Anemia Sideroblástica/etiologia , Animais , Heme/metabolismo , Heme/fisiologia , Hepatite C/etiologia , Humanos , Distúrbios do Metabolismo do Ferro/etiologia , Proteínas Ferro-Enxofre/biossíntese , Proteínas Ferro-Enxofre/fisiologia , Mitocôndrias/metabolismo , Doenças Neurodegenerativas/etiologia , Estresse OxidativoRESUMO
Aft1p is an iron-responsive transcriptional activator that plays a central role in maintaining iron homeostasis in Saccharomyces cerevisiae. Aft1p is regulated primarily by iron-induced shuttling of the protein between the nucleus and cytoplasm, but its nuclear import is not regulated by iron. Here, we have shown that the nuclear export of Aft1p is promoted in the presence of iron and that Msn5p is the nuclear export receptor (exportin) for Aft1p. Msn5p recognizes Aft1p in the iron-replete condition. Phosphorylation of S210 and S224 in Aft1p, which is not iron dependent, and the iron-induced intermolecular interaction of Aft1p are both essential for its recognition by Msn5p. Mutation of Cys291 of Aft1p to Phe, which causes Aft1p to be retained in the nucleus and results in constitutive activation of Aft1-target genes, disrupts the intermolecular interaction of Aft1p. Collectively, these results suggest that iron induces a conformational change in Aft1p, in which Aft1p Cys291 plays a critical role, and that, in turn, Aft1p is recognized by Msn5p and exported into the cytoplasm in an iron-dependent manner.
Assuntos
Núcleo Celular/metabolismo , Ferro/farmacologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Sequência de Aminoácidos , Núcleo Celular/efeitos dos fármacos , Carioferinas/metabolismo , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/efeitos dos fármacos , Proteínas de Saccharomyces cerevisiae/química , Serina , Treonina , Transativadores/metabolismo , Fatores de Transcrição/químicaRESUMO
Accurate chromosome segregation requires the execution and coordination of many processes during mitosis, including DNA replication, sister chromatid cohesion, and attachment of chromosomes to spindle microtubules via the kinetochore complex. Additional pathways are likely involved because faithful chromosome segregation also requires proteins that are not physically associated with the chromosome. Using kinetochore mutants as a starting point, we have identified genes with roles in chromosome stability by performing genome-wide screens employing synthetic genetic array methodology. Two genetic approaches (a series of synthetic lethal and synthetic dosage lethal screens) isolated 211 nonessential deletion mutants that were unable to tolerate defects in kinetochore function. Although synthetic lethality and synthetic dosage lethality are thought to be based upon similar genetic principles, we found that the majority of interactions associated with these two screens were nonoverlapping. To functionally characterize genes isolated in our screens, a secondary screen was performed to assess defects in chromosome segregation. Genes identified in the secondary screen were enriched for genes with known roles in chromosome segregation. We also uncovered genes with diverse functions, such as RCS1, which encodes an iron transcription factor. RCS1 was one of a small group of genes identified in all three screens, and we used genetic and cell biological assays to confirm that it is required for chromosome stability. Our study shows that systematic genetic screens are a powerful means to discover roles for uncharacterized genes and genes with alternative functions in chromosome maintenance that may not be discovered by using proteomics approaches.
Assuntos
Segregação de Cromossomos/genética , Genes Fúngicos , Genes Letais , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Saccharomyces cerevisiae/genética , Fatores de Transcrição de Zíper de Leucina e Hélice-Alça-Hélix Básicos/metabolismo , Instabilidade Cromossômica/genética , Cromossomos Fúngicos/metabolismo , Genômica/métodos , Cinetocoros/metabolismo , Mutação , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/análise , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/análise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
In Saccharomyces cerevisiae, the iron-responsive transcription factor Aft1p plays a critical role in maintaining iron homeostasis. The activity of Aft1p is induced in response to iron starvation and as a consequence the expression of the iron-regulon is increased. We have shown previously that Aft1p is localized to the cytoplasm under iron-replete conditions but that it is localized to the nucleus under iron-depleted conditions. In this study, we identified the transport receptor that mediates the import of Aft1p into the nucleus, located the nuclear localization signal (NLS) sequences of Aft1p, and examined whether the nuclear import of Aft1p is affected by iron status. In pse1-1 cells, which bear a temperature-sensitive mutation of PSE1, Aft1p was misdirected to the cytoplasm during iron starvation at the restrictive temperature. Aft1p could also directly bind to Pse1p and was dissociated from the complex by Ran-GTP in vitro. These results indicate that Aft1p is imported into the nucleus by Pse1p. Supporting this is that the induction of an Aft1p target gene, FTR1, in response to iron starvation was greatly reduced in pse1-1 cells. Furthermore, we demonstrated that the nuclear localization of a mutant Aft1 protein that contains an NLS derived from SV40 was regulated by iron status regardless of whether Pse1p could interact with Aft1p. This suggests that the interaction between Aft1p and Pse1p is not a critical step that controls the iron-regulated nucleo-cytoplasmic transport of Aft1p.
Assuntos
Transporte Ativo do Núcleo Celular , Ferro/metabolismo , Proteínas de Membrana Transportadoras , Receptores Citoplasmáticos e Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/fisiologia , Sequência de Aminoácidos , Northern Blotting , Núcleo Celular/metabolismo , Citoplasma/metabolismo , Técnica Indireta de Fluorescência para Anticorpo , Proteínas de Fluorescência Verde , Guanosina Trifosfato/metabolismo , Proteínas Luminescentes/metabolismo , Modelos Genéticos , Dados de Sequência Molecular , Sinais de Localização Nuclear , Plasmídeos/metabolismo , Ligação Proteica , RNA/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Temperatura , Fatores de Transcrição/metabolismo , Proteína ran de Ligação ao GTP/metabolismoRESUMO
The Aft1 transcription factor regulates the iron regulon in response to iron availability in Saccharomyces cerevisiae. Aft1 activates a battery of genes required for iron uptake under iron-starved conditions, whereas Aft1 function is inactivated under iron-replete conditions. Previously, we have shown that iron-regulated DNA binding by Aft1 is responsible for the controlled expression of target genes. Here we show that this iron-regulated DNA binding by Aft1 is not due to the change in the total expression level of Aft1 or alteration of DNA binding activity. Rather, nuclear localization of Aft1 responds to iron status, leading to iron-regulated expression of the target genes. We identified the nuclear export signal (NES)-like sequence in the AFT1 open reading frame. Mutation of the NES-like sequence causes nuclear retention of Aft1 and the constitutive activation of Aft1 function independent of the iron status of the cells. These results suggest that the nuclear export of Aft1 is critical for ensuring iron-responsive transcriptional activation of the Aft1 regulon and that the nuclear import/export systems are involved in iron sensing by Aft1 in S. cerevisiae.