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1.
PLoS One ; 15(3): e0221006, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32187190

RESUMO

Homeodomain-interacting protein kinases (Hipks) have been previously associated with cell proliferation and cancer, however, their effects in the nervous system are less well understood. We have used Drosophila melanogaster to evaluate the effects of altered Hipk expression on the nervous system and muscle. Using genetic manipulation of Hipk expression we demonstrate that knockdown and over-expression of Hipk produces early adult lethality, possibly due to the effects on the nervous system and muscle involvement. We find that optimal levels of Hipk are critical for the function of dopaminergic neurons and glial cells in the nervous system, as well as muscle. Furthermore, manipulation of Hipk affects the structure of the larval neuromuscular junction (NMJ) by promoting its growth. Hipk regulates the phosphorylation of the synapse-associated cytoskeletal protein Hu-li tai shao (Hts; adducin in mammals) and modulates the expression of two important protein kinases, Calcium-calmodulin protein kinase II (CaMKII) and Partitioning-defective 1 (PAR-1), all of which may alter neuromuscular structure/function and influence lethality. Hipk also modifies the levels of an important nuclear protein, TBPH, the fly orthologue of TAR DNA-binding protein 43 (TDP-43), which may have relevance for understanding motor neuron diseases.


Assuntos
Proteínas de Drosophila/isolamento & purificação , Drosophila melanogaster/enzimologia , Drosophila melanogaster/fisiologia , Músculos/anatomia & histologia , Músculos/metabolismo , Sistema Nervoso/anatomia & histologia , Sistema Nervoso/metabolismo , Proteínas Quinases/isolamento & purificação , Animais , Padronização Corporal , Núcleo Celular/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Olho/embriologia , Larva/metabolismo , Masculino , Músculos/citologia , Sistema Nervoso/citologia , Junção Neuromuscular/metabolismo , Tamanho do Órgão , Fosforilação , Sinapses/metabolismo
2.
PLoS One ; 15(2): e0228348, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32032373

RESUMO

The synaptic vesicle protein, synaptotagmin, is the principle Ca2+ sensor for synaptic transmission. Ca2+ influx into active nerve terminals is translated into neurotransmitter release by Ca2+ binding to synaptotagmin's tandem C2 domains, triggering the fast, synchronous fusion of multiple synaptic vesicles. Two hydrophobic residues, shown to mediate Ca2+-dependent membrane insertion of these C2 domains, are required for this process. Previous research suggested that one of its tandem C2 domains (C2B) is critical for fusion, while the other domain (C2A) plays only a facilitatory role. However, the function of the two hydrophobic residues in C2A have not been adequately tested in vivo. Here we show that these two hydrophobic residues are absolutely required for synaptotagmin to trigger vesicle fusion. Using in vivo electrophysiological recording at the Drosophila larval neuromuscular junction, we found that mutation of these two key C2A hydrophobic residues almost completely abolished neurotransmitter release. Significantly, mutation of both hydrophobic residues resulted in more severe deficits than those seen in synaptotagmin null mutants. Thus, we report the most severe phenotype of a C2A mutation to date, demonstrating that the C2A domain is absolutely essential for synaptotagmin's function as the electrostatic switch.


Assuntos
Cálcio/metabolismo , Proteínas de Drosophila/metabolismo , Transmissão Sináptica , Sinaptotagminas/metabolismo , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados/metabolismo , Drosophila/crescimento & desenvolvimento , Drosophila/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Potenciais Pós-Sinápticos Excitadores , Humanos , Larva/metabolismo , Larva/fisiologia , Mutagênese Sítio-Dirigida , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Sinaptotagminas/química , Sinaptotagminas/genética
3.
Nihon Saikingaku Zasshi ; 74(3): 167-175, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31787706

RESUMO

Botulinum neurotoxins (BoNTs) produced by the anaerobic bacterium Clostridium botulinum and related species cause botulism, a neuroparalytic disease associated with a high mortality. BoNTs are always produced as large protein complexes (progenitor toxin complexes, PTCs) through association with non-toxic components (NAPs) including hemagglutinin (HA) and non-toxic non-hemagglutinin (NTNHA). Food-borne botulism is caused by the ingestion of PTCs. PTCs in the gastrointestinal tract cross the intestinal epithelial barrier, enter the blood stream, and reach the nerve endings, where BoNTs cleave the SNAREs required for vesicle fusion. Consequently, BoNTs inhibit neurotransmitter release and cause paralysis. To cause food-borne botulism, BoNTs must traverse the intestinal epithelial barrier. However, the mechanism used to cross this barrier remains unclear. Using an in vitro epithelial barrier system, we previously showed that the interaction of HA with E-cadherin results in disruption of tight junctions. Furthermore, we previously reported that microfold (M) cells in the follicle-associated epithelium (FAE) of mouse Peyer's patches (PPs) are major sites where type A1 BoNT breaches the intestinal epithelial barrier. Here, I would like to demonstrate an ingenious invasion mechanism of the BoNT complex.


Assuntos
Toxinas Botulínicas/metabolismo , Células Epiteliais/metabolismo , Absorção Intestinal , Mucosa Intestinal/citologia , Complexos Multiproteicos/metabolismo , Neurotoxinas/metabolismo , Animais , Caderinas , Células Cultivadas , Cães , Hemaglutininas , Humanos , Camundongos , Terminações Nervosas/metabolismo , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Nódulos Linfáticos Agregados/metabolismo , Proteínas SNARE/metabolismo
4.
Nat Neurosci ; 22(11): 1793-1805, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31591561

RESUMO

Neuromuscular junction (NMJ) disruption is an early pathogenic event in amyotrophic lateral sclerosis (ALS). Yet, direct links between NMJ pathways and ALS-associated genes such as FUS, whose heterozygous mutations cause aggressive forms of ALS, remain elusive. In a knock-in Fus-ALS mouse model, we identified postsynaptic NMJ defects in newborn homozygous mutants that were attributable to mutant FUS toxicity in skeletal muscle. Adult heterozygous knock-in mice displayed smaller neuromuscular endplates that denervated before motor neuron loss, which is consistent with 'dying-back' neuronopathy. FUS was enriched in subsynaptic myonuclei, and this innervation-dependent enrichment was distorted in FUS-ALS. Mechanistically, FUS collaborates with the ETS transcription factor ERM to stimulate transcription of acetylcholine receptor genes. Co-cultures of induced pluripotent stem cell-derived motor neurons and myotubes from patients with FUS-ALS revealed endplate maturation defects due to intrinsic FUS toxicity in both motor neurons and myotubes. Thus, FUS regulates acetylcholine receptor gene expression in subsynaptic myonuclei, and muscle-intrinsic toxicity of ALS mutant FUS may contribute to dying-back motor neuronopathy.


Assuntos
Esclerose Amiotrófica Lateral/fisiopatologia , Regulação da Expressão Gênica/fisiologia , Degeneração Neural/fisiopatologia , Junção Neuromuscular/metabolismo , Proteína FUS de Ligação a RNA/fisiologia , Adulto , Esclerose Amiotrófica Lateral/patologia , Animais , Células Cultivadas , Feminino , Técnicas de Introdução de Genes , Humanos , Masculino , Camundongos , Camundongos Knockout , Neurônios Motores/patologia , Fibras Musculares Esqueléticas/patologia , Junção Neuromuscular/patologia , Proteína FUS de Ligação a RNA/genética , Proteína FUS de Ligação a RNA/metabolismo , Receptores Colinérgicos/metabolismo , Adulto Jovem
5.
Int J Mol Sci ; 20(19)2019 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-31574943

RESUMO

Lipid raft disruption is an early event during skeletal muscle unloading. Ceramide (Cer) serves as a signaling lipid that can contribute to lipid raft disturbance and muscle atrophy. Using biochemical and fluorescent approaches, the distribution of Cer and related molecules in the rat soleus muscle subjected to 12 h of hindlimb suspension (HS) was studied. HS led to upregulation of TNFα receptor 1 (TNFR1), Cer-producing enzymes, and acid and neutral sphingomyelinase (SMase) in detergent-resistant membranes (lipid rafts), which was accompanied by an increase in Cer and a decrease in sphingomyelin in this membrane fraction. Fluorescent labeling indicated increased Cer in the sarcoplasm as well as the junctional (synaptic) and extrajunctional compartments of the suspended muscles. Also, a loss of membrane asymmetry (a hallmark of membrane disturbance) was induced by HS. Pretreatment with clomipramine, a functional inhibitor of acid SMase, counteracted HS-mediated changes in the Cer/sphingomyelin ratio and acid SMase abundance as well as suppressed Cer accumulation in the intracellular membranes of junctional and extrajunctional regions. However, the elevation of plasma membrane Cer and disturbance of the membrane asymmetry were suppressed only in the junctional compartment. We suggest that acute HS leads to TNFR1 and SMase upregulation in the lipid raft fraction and deposition of Cer throughout the sarcolemma and intracellularly. Clomipramine-mediated downregulation of acid SMase can suppress Cer accumulation in all compartments, excluding the extrajunctional plasma membrane.


Assuntos
Membrana Celular/metabolismo , Ceramidas/metabolismo , Músculo Esquelético/metabolismo , Animais , Biomarcadores , Imunofluorescência , Microdomínios da Membrana/metabolismo , Junção Neuromuscular/metabolismo , Ratos , Esfingomielina Fosfodiesterase/metabolismo
6.
Muscle Nerve ; 60(6): 790-800, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31531871

RESUMO

INTRODUCTION: Reduced expression of the vesicular acetylcholine transporter (VAChT) leads to changes in the distribution and shape of synaptic vesicles (SVs) at neuromuscular junctions (NMJs), suggesting vesicular acetylcholine (ACh) as a key component of synaptic structure and function. It is poorly understood how long-term changes in cholinergic transmission contribute to age- and disease-related degeneration in the motor system. METHODS: In this study we performed confocal imaging, electrophysiology, electron microscopy, and analyses of respiratory mechanics of the diaphragm NMJ components in 12-month-old wild-type (WT) and VAChTKDHOM mice. RESULTS: Diaphragms of NMJs of the VAChTKDHOM mice were similar to those in WT mice in number, colocalization, and fragmentation of pre-/postsynaptic components. However, they had increased spontaneous SV exocytosis, miniature endplate potential frequency, and diminished MEPP amplitude. No impairment in respiratory mechanics at rest was observed, probably due to the large neurotransmission safety factor of the diaphragm. DISCUSSION: The present findings help us to understand the consequences of reduced ACh release at the NMJs during aging.


Assuntos
Envelhecimento/patologia , Diafragma/ultraestrutura , Síndromes Miastênicas Congênitas/patologia , Junção Neuromuscular/ultraestrutura , Vesículas Sinápticas/ultraestrutura , Acetilcolina/metabolismo , Envelhecimento/metabolismo , Animais , Diafragma/metabolismo , Diafragma/fisiopatologia , Modelos Animais de Doenças , Endocitose , Potenciais Pós-Sinápticos Excitadores/fisiologia , Exocitose , Técnicas de Silenciamento de Genes , Camundongos , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Placa Motora , Síndromes Miastênicas Congênitas/genética , Síndromes Miastênicas Congênitas/metabolismo , Síndromes Miastênicas Congênitas/fisiopatologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/fisiopatologia , Mecânica Respiratória/fisiologia , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Proteínas Vesiculares de Transporte de Acetilcolina/genética
7.
PLoS Genet ; 15(8): e1007980, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31381576

RESUMO

Synaptic structure and activity are sensitive to environmental alterations. Modulation of synaptic morphology and function is often induced by signals from glia. However, the process by which glia mediate synaptic responses to environmental perturbations such as hypoxia remains unknown. Here, we report that, in the mutant for Trachealess (Trh), the Drosophila homolog for NPAS1 and NPAS3, smaller synaptic boutons form clusters named satellite boutons appear at larval neuromuscular junctions (NMJs), which is induced by the reduction of internal oxygen levels due to defective tracheal branches. Thus, the satellite bouton phenotype in the trh mutant is suppressed by hyperoxia, and recapitulated in wild-type larvae raised under hypoxia. We further show that hypoxia-inducible factor (HIF)-1α/Similar (Sima) is critical in mediating hypoxia-induced satellite bouton formation. Sima upregulates the level of the Wnt/Wingless (Wg) signal in glia, leading to reorganized microtubule structures within presynaptic sites. Finally, hypoxia-induced satellite boutons maintain normal synaptic transmission at the NMJs, which is crucial for coordinated larval locomotion.


Assuntos
Hipóxia Celular/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/metabolismo , Junção Neuromuscular/crescimento & desenvolvimento , Transmissão Sináptica/fisiologia , Fatores de Transcrição/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Microscopia Intravital , Larva/fisiologia , Locomoção/genética , Microscopia Confocal , Microtúbulos/metabolismo , Modelos Animais , Neuroglia/citologia , Neuroglia/metabolismo , Junção Neuromuscular/metabolismo , Terminações Pré-Sinápticas/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Regulação para Cima , Proteína Wnt1/metabolismo
8.
Exp Parasitol ; 205: 107734, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31394093

RESUMO

Parasitism by Haemonchus contortus is one of the main limiting factors in small ruminant production around the globe. Although several studies suggest the use of integrated management practices, these parasites have been controlled essentially with synthetic anthelmintic drugs. The resistance mechanism against the imidazothiazole derivative levamisole in Haemonchus contortus has not been fully described. Recently, resistance was associated with a 63bp deletion in the Hco-acr-8b gene that encodes a subunit for a nicotinic acetylcholine receptor. This study aimed to standardize a real time PCR (qPCR) protocol for levamisole resistance diagnosis in H. contortus populations based on this polymorphism and use it to characterize 23 field H. contortus populations obtained from different localities of Ceará State, Northeast Brazil. In addition, two populations of H. contortus were used as a standard of susceptibility and resistance, Inbred Strain Edinburgh (ISE) and Kokstad, respectively. Larval development tests (LDT) were performed on five field isolates and both EC50 and EC95 were estimated. LDT EC95 values provided a wider interval between susceptible and resistant populations than EC50 values (EC95 = 1.96-57.93 µM; EC50 = 0.05-0.39 µM), and were found to be more appropriate for differentiating them. Real time PCR results showed resistance allele frequencies ranged from 20.9 to 76.7%. Our results suggest that levamisole resistance may be present in field populations but it is not as widespread as benzimidazole resistance. This methodology may be useful to monitor levamisole resistance in field populations of H. contortus.


Assuntos
Antinematódeos/farmacologia , Resistência a Medicamentos/genética , Haemonchus/efeitos dos fármacos , Levamisol/farmacologia , Animais , Benzimidazóis/farmacologia , DNA de Helmintos/isolamento & purificação , Fezes/parasitologia , Frequência do Gene/genética , Hemoncose/tratamento farmacológico , Hemoncose/parasitologia , Hemoncose/veterinária , Haemonchus/genética , Haemonchus/crescimento & desenvolvimento , Larva/efeitos dos fármacos , Larva/crescimento & desenvolvimento , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/veterinária , Receptores Colinérgicos/efeitos dos fármacos , Receptores Nicotínicos/efeitos dos fármacos , Alinhamento de Sequência/veterinária , Ovinos , Doenças dos Ovinos/tratamento farmacológico , Doenças dos Ovinos/parasitologia , Tetramizol/farmacologia
9.
Handb Clin Neurol ; 160: 3-22, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31277855

RESUMO

The action potential is a regenerative electrical phenomenon observed on excitable cell membranes that allows the propagation of signals without attenuation. It is the cornerstone of neurophysiology. This chapter is a review of the action potential and its relationship to the signals that are studied in clinical neurophysiology. The first section traces the history of key scientific discoveries over the last 250 years that have led to our present-day understanding of the electrical properties of nerve and muscle. The second section considers the molecular and biophysical mechanisms that are responsible for the electrical potentials that can be measured across all eukaryotic cell membranes, but specifically in neurons, nerves, and muscle. Mechanisms underlying propagation action potentials within the nervous system are also examined. The concluding section is a brief overview of the normal "macroscopic" signals that are commonly recorded from the central and peripheral nervous system, and how they are derived from action potentials.


Assuntos
Potenciais de Ação/fisiologia , Neurônios Motores/fisiologia , Condução Nervosa/fisiologia , Junção Neuromuscular/metabolismo , Animais , Eletromiografia/métodos , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Recrutamento Neurofisiológico/fisiologia
10.
Elife ; 82019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31318331

RESUMO

Overproduction of reactive oxygen species (ROS) is known to mediate glutamate excitotoxicity in neurological diseases. However, how ROS burdens can influence neural circuit integrity remains unclear. Here, we investigate the impact of excitotoxicity induced by depletion of Drosophila Eaat1, an astrocytic glutamate transporter, on locomotor central pattern generator (CPG) activity, neuromuscular junction architecture, and motor function. We show that glutamate excitotoxicity triggers a circuit-dependent ROS feedback loop to sculpt the motor system. Excitotoxicity initially elevates ROS, thereby inactivating cholinergic interneurons and consequently changing CPG output activity to overexcite motor neurons and muscles. Remarkably, tonic motor neuron stimulation boosts muscular ROS, gradually dampening muscle contractility to feedback-enhance ROS accumulation in the CPG circuit and subsequently exacerbate circuit dysfunction. Ultimately, excess premotor excitation of motor neurons promotes ROS-activated stress signaling that alters neuromuscular junction architecture. Collectively, our results reveal that excitotoxicity-induced ROS can perturb motor system integrity through a circuit-dependent mechanism.


Assuntos
Drosophila melanogaster/fisiologia , Retroalimentação Fisiológica , Ácido Glutâmico/toxicidade , Neurônios Motores/fisiologia , Neurotoxinas/toxicidade , Espécies Reativas de Oxigênio/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Neurônios Colinérgicos/efeitos dos fármacos , Neurônios Colinérgicos/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Interneurônios/efeitos dos fármacos , Interneurônios/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Mutação/genética , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Estresse Oxidativo/efeitos dos fármacos
11.
Nat Commun ; 10(1): 2998, 2019 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-31278365

RESUMO

At the Drosophila neuromuscular junction, inhibition of postsynaptic glutamate receptors activates retrograde signaling that precisely increases presynaptic neurotransmitter release to restore baseline synaptic strength. However, the nature of the underlying postsynaptic induction process remains enigmatic. Here, we design a forward genetic screen to discover factors in the postsynaptic compartment necessary to generate retrograde homeostatic signaling. This approach identified insomniac (inc), a putative adaptor for the Cullin-3 (Cul3) ubiquitin ligase complex, which together with Cul3 is essential for normal sleep regulation. Interestingly, we find that Inc and Cul3 rapidly accumulate at postsynaptic compartments following acute receptor inhibition and are required for a local increase in mono-ubiquitination. Finally, we show that Peflin, a Ca2+-regulated Cul3 co-adaptor, is necessary for homeostatic communication, suggesting a relationship between Ca2+ signaling and control of Cul3/Inc activity in the postsynaptic compartment. Our study suggests that Cul3/Inc-dependent mono-ubiquitination, compartmentalized at postsynaptic densities, gates retrograde signaling and provides an intriguing molecular link between the control of sleep and homeostatic plasticity at synapses.


Assuntos
Proteínas Culina/metabolismo , Proteínas de Drosophila/metabolismo , Somação de Potenciais Pós-Sinápticos/fisiologia , Terminações Pré-Sinápticas/metabolismo , Sono/fisiologia , Animais , Drosophila melanogaster , Feminino , Homeostase/fisiologia , Masculino , Modelos Animais , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Ubiquitinação/fisiologia
13.
Int J Mol Sci ; 20(11)2019 Jun 08.
Artigo em Inglês | MEDLINE | ID: mdl-31181747

RESUMO

Neuromuscular junction (NMJ) formation involves morphological changes both in motor terminals and muscle membrane. The molecular mechanisms leading to NMJ formation and maintenance have not yet been fully elucidated. During the last decade, it has become clear that virtually all cells release different types of extracellular vesicles (EVs), which can be taken up by nearby or distant cells modulating their activity. Initially, EVs were associated to a mechanism involved in the elimination of unwanted material; subsequent evidence demonstrated that exosomes, and more in general EVs, play a key role in intercellular communication by transferring proteins, lipids, DNA and RNA to target cells. Recently, EVs have emerged as potent carriers for Wnt, bone morphogenetic protein, miRNA secretion and extracellular traveling. Convincing evidence demonstrates that presynaptic terminals release exosomes that are taken up by muscle cells, and these exosomes can modulate synaptic plasticity in the recipient muscle cell in vivo. Furthermore, recent data highlighted that EVs could also be a potential cause of neurodegenerative disorders. Indeed, mutant SOD1, TDP-43 and FUS/TLS can be secreted by neural cells packaged into EVs and enter in neighboring neural cells, contributing to the onset and severity of the disease.


Assuntos
Vesículas Extracelulares/metabolismo , Junção Neuromuscular/metabolismo , Transdução de Sinais , Animais , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Doença dos Neurônios Motores/etiologia , Neurogênese , Junção Neuromuscular/citologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiologia
14.
Int J Mol Sci ; 20(11)2019 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-31159267

RESUMO

Synaptic vesicles dock on the presynaptic plasma membrane of axon terminals and become ready to fuse with the presynaptic membrane or primed. Fusion of the vesicle membrane and presynaptic membrane results in the formation of a pore between the membranes, through which the vesicle's neurotransmitter is released into the synaptic cleft. A recent electron tomography study on frog neuromuscular junctions fixed at rest showed that there is no discernible gap between or merging of the membrane of docked synaptic vesicles with the presynaptic membrane, however, the extent of the contact area between the membrane of docked synaptic vesicles and the presynaptic membrane varies 10-fold with a normal distribution. The study also showed that when the neuromuscular junctions are fixed during repetitive electrical nerve stimulation, the portion of large contact areas in the distribution is reduced compared to the portion of small contact areas, suggesting that docked synaptic vesicles with the largest contact areas are greatly primed to fuse with the membrane. Furthermore, the finding of several hemifused synaptic vesicles among the docked vesicles was briefly reported. Here, the spatial relationship of 81 synaptic vesicles with the presynaptic membrane at active zones of the neuromuscular junctions fixed during stimulation is described in detail. For the most of the vesicles, the combined thickness of each of their contact sites was not different from the sum of the membrane thicknesses of the vesicle membrane and presynaptic membrane, similar to the docked vesicles at active zones of the resting neuromuscular junctions. However, the combined membrane thickness of a small portion of the vesicles was considerably less than the sum of the membrane thicknesses, indicating that the membranes at their contact sites were fixed in a state of hemifusion. Moreover, the hemifused vesicles were found to have large contact areas with the presynaptic membrane. These findings support the recently proposed hypothesis that, at frog neuromuscular junctions, docked synaptic vesicles with the largest contact areas are most primed for fusion with the presynaptic membrane, and that hemifusion is a fusion intermediate step of the vesicle membrane with the presynaptic membrane for synaptic transmission.


Assuntos
Junção Neuromuscular/metabolismo , Membranas Sinápticas/metabolismo , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Animais , Anuros , Modelos Biológicos , Junção Neuromuscular/ultraestrutura , Membranas Sinápticas/ultraestrutura , Vesículas Sinápticas/ultraestrutura
15.
J Surg Res ; 241: 308-316, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31055156

RESUMO

BACKGROUND: Abnormal expression and distribution of nicotinic acetylcholine receptors (nAChRs) in skeletal muscle caused by sepsis can lead to neuromuscular dysfunction. Here, we asked whether neural agrin regulates nAChRs to ameliorate muscle function, which could be associated with the agrin/muscle-specific kinase pathway. METHODS: Rats were subjected to cecal ligation and puncture (CLP) group, sham group, or control group to observe the alteration caused by sepsis. To verify the effect of improving function, rats were injected with agrin or normal saline intramuscularly after CLP. Electromyogram was used to measure neuromuscular function. Cytokines levels of serum and the expression of related proteins and mRNA were tested after treatment. RESULTS: Compared with the rats in control or sham group, CLP-treated rats showed an acute inflammatory status and a reduction of neuromuscular dysfunction in tibialis anterior muscle, which was associated with abnormal expression in agrin/muscle-specific kinase pathway and increased expression of γ- and α7-nAChR. Exogenous agrin alleviated neuromuscular dysfunction and decreased the expression of γ- and α7-nAChR through agrin-related signaling pathway. CONCLUSIONS: The decreased expression of agrin may lead to skeletal muscle dysfunction. Early enhancement of intramuscular agrin levels after sepsis may be a potential strategy for the treatment of sepsis-induced muscle dysfunction.


Assuntos
Agrina/metabolismo , Doenças da Junção Neuromuscular/imunologia , Sepse/imunologia , Transdução de Sinais/imunologia , Receptor Nicotínico de Acetilcolina alfa7/metabolismo , Agrina/imunologia , Animais , Modelos Animais de Doenças , Humanos , Masculino , Músculo Esquelético/imunologia , Músculo Esquelético/inervação , Junção Neuromuscular/imunologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Doenças da Junção Neuromuscular/patologia , Ratos , Ratos Sprague-Dawley , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/imunologia , Receptores Colinérgicos/metabolismo , Sepse/complicações , Receptor Nicotínico de Acetilcolina alfa7/imunologia
16.
Mol Cell Biol ; 39(14)2019 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-31036568

RESUMO

GALGT2 (also B4GALNT2) encodes a glycosyltransferase that is normally confined to the neuromuscular and myotendinous junction in adult skeletal muscle. GALGT2 overexpression in muscle can inhibit muscular dystrophy in mouse models of the disease by inducing the overexpression of surrogate muscle proteins, including utrophin, agrin, laminins, and integrins. Despite its well-documented biological properties, little is known about the endogenous regulation of muscle GALGT2 expression. Here, we demonstrate that epidermal growth factor receptor (EGFR) ligands can activate the human GALGT2 promoter. Overexpression of one such ligand, soluble heparin-binding EGF-like growth factor (sHB-EGF), also stimulated mouse muscle Galgt2 gene expression and expression of GALGT2-inducible surrogate muscle genes. Deletion analysis of the GALGT2 promoter identified a 45-bp region containing a TFAP4-binding site that was required for sHB-EGF activation. sHB-EGF increased TFAP4 binding to this site in muscle cells and increased endogenous Tfap4 gene expression. sHB-EGF also increased muscle EGFR protein expression and activated EGFR-Akt signaling. sHB-EGF expression was concentrated at the neuromuscular junction, and Hbegf deletion reduced Galgt2-dependent synaptic glycosylation. Hbegf deletion also mimicked Galgt2-dependent neuromuscular and muscular dystrophy phenotypes. These data demonstrate that sHB-EGF is an endogenous regulator of muscle Galgt2 gene expression and can mimic Galgt2-dependent muscle phenotypes.


Assuntos
Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/metabolismo , Músculo Esquelético/metabolismo , N-Acetilgalactosaminiltransferases/genética , Junção Neuromuscular/metabolismo , Animais , Sítios de Ligação , Células CHO , Linhagem Celular , Cricetulus , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Glicosilação , Células HEK293 , Humanos , Masculino , Camundongos , N-Acetilgalactosaminiltransferases/química , N-Acetilgalactosaminiltransferases/metabolismo , Regiões Promotoras Genéticas , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
17.
Mol Neurobiol ; 56(10): 6856-6872, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30929165

RESUMO

Amyotrophic lateral sclerosis (ALS) is a chronic neurodegenerative disease characterized by progressive motor weakness. It is accepted that it is caused by motoneuron degeneration leading to a decrease in muscle stimulation. However, ALS is being redefined as a distal axonopathy, in that neuromuscular junction dysfunction precedes and may even influence motoneuron loss. In this synapse, several metabotropic receptor-mediated signaling pathways converge on effector kinases that phosphorylate targets that are crucial for synaptic stability and neurotransmission quality. We have previously shown that, in physiological conditions, nerve-induced muscle contraction regulates the brain-derived neurotrophic factor/tropomyosin-related kinase B (BDNF/TrkB) signaling to retrogradely modulate presynaptic protein kinases PKC and PKA, which are directly involved in the modulation of acetylcholine release. In ALS patients, the alteration of this signaling may significantly contribute to a motor impairment. Here, we investigate whether BDNF/TrkB signaling, the downstream PKC (cPKCßI, cPKCα, and nPKCε isoforms), and PKA (regulatory and catalytic subunits) and some SNARE/SM exocytotic machinery proteins (Munc18-1 and SNAP-25) are altered in the skeletal muscle of pre- and symptomatic SOD1-G93A mice. We found that this pathway is strongly affected in symptomatic ALS mice muscles including an unbalance between (I) BDNF and TrkB isoforms, (II) PKC isoforms and PKA subunits, and (III) Munc18-1 and SNAP-25 phosphorylation ratios. Changes in TrkB.T1 and cPKCßI are precociously observed in presymptomatic mice. Altogether, several of these molecular alterations can be partly associated with the known fast-to-slow motor unit transition during the disease process but others can be related with the initial disease pathogenesis.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Junção Neuromuscular/patologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas SNARE/metabolismo , Transdução de Sinais , Superóxido Dismutase-1/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Domínio Catalítico , Modelos Animais de Doenças , Masculino , Camundongos Transgênicos , Modelos Biológicos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Músculos/metabolismo , Músculos/patologia , Fatores de Crescimento Neural/metabolismo , Junção Neuromuscular/metabolismo , Receptores de Fator de Crescimento Neural/metabolismo , Medula Espinal/patologia
18.
Nat Commun ; 10(1): 1085, 2019 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-30842428

RESUMO

Neuronal communication across synapses relies on neurotransmitter release from presynaptic active zones (AZs) followed by postsynaptic transmitter detection. Synaptic plasticity homeostatically maintains functionality during perturbations and enables memory formation. Postsynaptic plasticity targets neurotransmitter receptors, but presynaptic mechanisms regulating the neurotransmitter release apparatus remain largely enigmatic. By studying Drosophila neuromuscular junctions (NMJs) we show that AZs consist of nano-modular release sites and identify a molecular sequence that adds modules within minutes of inducing homeostatic plasticity. This requires cognate transport machinery and specific AZ-scaffolding proteins. Structural remodeling is not required for immediate potentiation of neurotransmitter release, but necessary to sustain potentiation over longer timescales. Finally, mutations in Unc13 disrupting homeostatic plasticity at the NMJ also impair short-term memory when central neurons are targeted, suggesting that both plasticity mechanisms utilize Unc13. Together, while immediate synaptic potentiation capitalizes on available material, it triggers the coincident incorporation of modular release sites to consolidate synaptic potentiation.


Assuntos
Proteínas de Drosophila/metabolismo , Potenciação de Longa Duração/fisiologia , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Junção Neuromuscular/metabolismo , Neurotransmissores/metabolismo , Terminações Pré-Sinápticas/metabolismo , Animais , Animais Geneticamente Modificados , Comportamento Animal , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Feminino , Masculino , Proteínas de Membrana/genética , Memória de Curto Prazo/fisiologia , Modelos Animais , Corpos Pedunculados/citologia , Corpos Pedunculados/metabolismo , Proteínas do Tecido Nervoso/genética , Técnicas de Patch-Clamp , Transmissão Sináptica/fisiologia , Vesículas Sinápticas/metabolismo
19.
Int J Mol Sci ; 20(5)2019 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-30823359

RESUMO

A present review is devoted to the analysis of literature data and results of own research. Skeletal muscle neuromuscular junction is specialized to trigger the striated muscle fiber contraction in response to motor neuron activity. The safety factor at the neuromuscular junction strongly depends on a variety of pre- and postsynaptic factors. The review focuses on the crucial role of membrane cholesterol to maintain a high efficiency of neuromuscular transmission. Cholesterol metabolism in the neuromuscular junction, its role in the synaptic vesicle cycle and neurotransmitter release, endplate electrogenesis, as well as contribution of cholesterol to the synaptogenesis, synaptic integrity, and motor disorders are discussed.


Assuntos
Colesterol/metabolismo , Junção Neuromuscular/metabolismo , Transmissão Sináptica , Animais , Humanos , Junção Neuromuscular/fisiologia
20.
Genetics ; 212(1): 231-243, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30898771

RESUMO

Precise signaling at the neuromuscular junction (NMJ) is essential for proper muscle contraction. In the Caenorhabditis elegans pharynx, acetylcholine (ACh) released from the MC and M4 motor neurons stimulates two different types of contractions in adjacent muscle cells, termed pumping and isthmus peristalsis. MC stimulates rapid pumping through the nicotinic ACh receptor EAT-2, which is tightly localized at the MC NMJ, and eat-2 mutants exhibit a slow pump rate. Surprisingly, we found that eat-2 mutants also hyperstimulated peristaltic contractions, and that they were characterized by increased and prolonged Ca2+ transients in the isthmus muscles. This hyperstimulation depends on cross talk with the GAR-3 muscarinic ACh receptor as gar-3 mutation specifically suppressed the prolonged contraction and increased Ca2+ observed in eat-2 mutant peristalses. Similar GAR-3-dependent hyperstimulation was also observed in mutants lacking the ace-3 acetylcholinesterase, and we suggest that NMJ defects in eat-2 and ace-3 mutants result in ACh stimulation of extrasynaptic GAR-3 receptors in isthmus muscles. gar-3 mutation also suppressed slow larval growth and prolonged life span phenotypes that result from dietary restriction in eat-2 mutants, indicating that cross talk with the GAR-3 receptor has a long-term impact on feeding behavior and eat-2 mutant phenotypes.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Contração Muscular , Músculos/metabolismo , Junção Neuromuscular/metabolismo , Receptores Muscarínicos/metabolismo , Receptores Nicotínicos/metabolismo , Animais , Animais Geneticamente Modificados , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/fisiologia , Neurônios Motores , Músculos/fisiologia , Faringe/metabolismo , Faringe/fisiologia , Receptores Muscarínicos/fisiologia , Receptores Nicotínicos/fisiologia , Transdução de Sinais
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