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1.
Nucleic Acids Res ; 48(6): 2853-2865, 2020 04 06.
Artigo em Inglês | MEDLINE | ID: mdl-32103257

RESUMO

Spinal muscular atrophy (SMA) is a motor neuron disease. Nusinersen, a splice-switching antisense oligonucleotide (ASO), was the first approved drug to treat SMA. Based on prior preclinical studies, both 2'-O-methoxyethyl (MOE) with a phosphorothioate backbone and morpholino with a phosphorodiamidate backbone-with the same or extended target sequence as nusinersen-displayed efficient rescue of SMA mouse models. Here, we compared the therapeutic efficacy of these two modification chemistries in rescue of a severe mouse model using ASO10-29-a 2-nt longer version of nusinersen-via subcutaneous injection. Although both chemistries efficiently corrected SMN2 splicing in various tissues, restored motor function and improved the integrity of neuromuscular junctions, MOE-modified ASO10-29 (MOE10-29) was more efficacious than morpholino-modified ASO10-29 (PMO10-29) at the same molar dose, as seen by longer survival, greater body-weight gain and better preservation of motor neurons. Time-course analysis revealed that MOE10-29 had more persistent effects than PMO10-29. On the other hand, PMO10-29 appears to more readily cross an immature blood-brain barrier following systemic administration, showing more robust initial effects on SMN2 exon 7 inclusion, but less persistence in the central nervous system. We conclude that both modifications can be effective as splice-switching ASOs in the context of SMA and potentially other diseases, and discuss the advantages and disadvantages of each.


Assuntos
Amidas/química , Morfolinos/uso terapêutico , Atrofia Muscular Espinal/tratamento farmacológico , Oligonucleotídeos Antissenso/uso terapêutico , Ácidos Fosfóricos/química , Animais , Modelos Animais de Doenças , Éxons/genética , Humanos , Camundongos Transgênicos , Morfolinos/farmacologia , Atividade Motora/efeitos dos fármacos , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Músculos/patologia , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/fisiopatologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Oligonucleotídeos Antissenso/farmacologia , Fenótipo , Processamento de RNA/efeitos dos fármacos , Processamento de RNA/genética , Medula Espinal/patologia , Proteína 2 de Sobrevivência do Neurônio Motor/genética , Resultado do Tratamento
2.
Nat Protoc ; 15(2): 421-449, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932771

RESUMO

This protocol describes the design, fabrication and use of a 3D physiological and pathophysiological motor unit model consisting of motor neurons coupled to skeletal muscles interacting via the neuromuscular junction (NMJ) within a microfluidic device. This model facilitates imaging and quantitative functional assessment. The 'NMJ chip' enables real-time, live imaging of axonal outgrowth, NMJ formation and muscle maturation, as well as synchronization of motor neuron activity and muscle contraction under optogenetic control for the study of normal physiological events. The proposed protocol takes ~2-3 months to be implemented. Pathological behaviors associated with various neuromuscular diseases, such as regression of motor neuron axons, motor neuron death, and muscle degradation and atrophy can also be recapitulated in this system. Disease models can be created by the use of patient-derived induced pluripotent stem cells to generate both the motor neurons and skeletal muscle cells used. This is demonstrated by the use of cells from a patient with sporadic amyotrophic lateral sclerosis but can be applied more generally to models of neuromuscular disease, such as spinal muscular atrophy, NMJ disorder and muscular dystrophy. Models such as this hold considerable potential for applications in precision medicine, drug screening and disease risk assessment.


Assuntos
Avaliação Pré-Clínica de Medicamentos/instrumentação , Procedimentos Analíticos em Microchip/métodos , Doenças Neuromusculares/tratamento farmacológico , Medicina de Precisão/instrumentação , Humanos , Contração Muscular/efeitos dos fármacos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Doenças Neuromusculares/patologia , Doenças Neuromusculares/fisiopatologia , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Medição de Risco
3.
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
4.
Cells ; 8(8)2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31426366

RESUMO

One of the crucial systems severely affected in several neuromuscular diseases is the loss of effective connection between muscle and nerve, leading to a pathological non-communication between the two tissues. The neuromuscular junction (NMJ) represents the critical region at the level of which muscle and nerve communicate. Defects in signal transmission between terminal nerve endings and muscle membrane is a common feature of several physio-pathologic conditions including aging and Amyotrophic Lateral Sclerosis (ALS). Nevertheless, controversy exists on whether pathological events beginning at the NMJ precede or follow loss of motor units. In this review, the role of NMJ in the physio-pathologic interplay between muscle and nerve is discussed.


Assuntos
Envelhecimento/patologia , Esclerose Amiotrófica Lateral/patologia , Junção Neuromuscular/patologia , Proteína Quinase C/metabolismo , Células de Schwann , Superóxido Dismutase-1/metabolismo , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Células de Schwann/metabolismo , Células de Schwann/patologia
5.
Muscle Nerve ; 60(5): 604-612, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31408210

RESUMO

INTRODUCTION: In this study we present a reproducible technique to assess motor recovery after nerve injury via neuromuscular junction (NMJ) immunostaining and electrodiagnostic testing. METHODS: Wild-type mice underwent sciatic nerve transection with repair. Hindlimb muscles were collected for microscopy up to 30 weeks after injury. Immunostaining was used to assess axons (NF200), Schwann cells (S100), and motor endplates (α-bungarotoxin). Compound motor action potential (CMAP) amplitude was used to assess tibialis anterior (TA) function. RESULTS: One week after injury, nearly all (98.0%) endplates were denervated. At 8 weeks, endplates were either partially (28.3%) or fully (71.7%) reinnervated. At 16 weeks, NMJ reinnervation reached 87.3%. CMAP amplitude was 83% of naive mice at 16 weeks and correlated with percentage of fully reinnervated NMJs. Morphological differences were noted between injured and noninjured NMJs. DISCUSSION: We present a reproducible method for evaluating NMJ reinnervation. Electrodiagnostic data summarize NMJ recovery. Characterization of wild-type reinnervation provides important data for consideration in experimental design and interpretation.


Assuntos
Potenciais de Ação/fisiologia , Axônios/patologia , Músculo Esquelético/inervação , Regeneração Nervosa/fisiologia , Junção Neuromuscular/patologia , Células de Schwann/patologia , Animais , Bungarotoxinas , Camundongos , Placa Motora/patologia , Placa Motora/fisiopatologia , Denervação Muscular , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Proteínas de Neurofilamentos , Junção Neuromuscular/fisiopatologia , Procedimentos Neurocirúrgicos , Recuperação de Função Fisiológica , Proteínas S100 , Nervo Isquiático/lesões , Nervo Isquiático/cirurgia , Coloração e Rotulagem , Cicatrização
6.
Cells ; 8(7)2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31269763

RESUMO

Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ). Autoantibodies target key molecules at the NMJ, such as the nicotinic acetylcholine receptor (AChR), muscle-specific kinase (MuSK), and low-density lipoprotein receptor-related protein 4 (Lrp4), that lead by a range of different pathogenic mechanisms to altered tissue architecture and reduced densities or functionality of AChRs, reduced neuromuscular transmission, and therefore a severe fatigable skeletal muscle weakness. In this review, we give an overview of the history and clinical aspects of MG, with a focus on the structure and function of myasthenic autoantigens at the NMJ and how they are affected by the autoantibodies' pathogenic mechanisms. Furthermore, we give a short overview of the cells that are implicated in the production of the autoantibodies and briefly discuss diagnostic challenges and treatment strategies.


Assuntos
Autoanticorpos/imunologia , Autoantígenos/imunologia , Músculo Esquelético/patologia , Miastenia Gravis/imunologia , Junção Neuromuscular/patologia , Agrina/imunologia , Agrina/metabolismo , Animais , Autoantígenos/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL/imunologia , Proteínas Relacionadas a Receptor de LDL/metabolismo , Músculo Esquelético/imunologia , Músculo Esquelético/ultraestrutura , Miastenia Gravis/patologia , Junção Neuromuscular/imunologia , Junção Neuromuscular/ultraestrutura , Receptores Proteína Tirosina Quinases/imunologia , Receptores Proteína Tirosina Quinases/metabolismo , Receptores Colinérgicos/imunologia , Receptores Colinérgicos/metabolismo , Receptores Nicotínicos/imunologia , Receptores Nicotínicos/metabolismo
7.
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
8.
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
9.
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
10.
Restor Neurol Neurosci ; 37(2): 181-196, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31006701

RESUMO

BACKGROUND: The "post-paralytic syndrome" after facial nerve reconstruction has been attributed to (i) malfunctioning axonal guidance at the fascicular (branches) level, (ii) collateral branching of the transected axons at the lesion site, and (iii) intensive intramuscular terminal sprouting of regenerating axons which causes poly-innervation of the neuromuscular junctions (NMJ). OBJECTIVE: The first two reasons were approached by an innovative technique which should provide the re-growing axons optimal conditions to elongate and selectively re-innervate their original muscle groups. METHODS: The transected facial nerve trunk was inserted into a 3-way-conduit (from isogeneic rat abdominal aorta) which should "guide" the re-growing facial axons to the three main branches of the facial nerve (zygomatic, buccal and marginal mandibular). The effect of this method was tested also on hypoglossal axons after hypoglossal-facial anastomosis (HFA). Coaptational (classic) FFA (facial-facial anastomosis) and HFA served as controls. RESULTS: When compared to their coaptation (classic) alternatives, both types of 3-way-conduit operations (FFA and HFA) promoted a trend for reduction in the collateral axonal branching (the proportion of double- or triple-labelled perikarya after retrograde tracing was slightly reduced). In contrast, poly-innervation of NMJ in the levator labii superioris muscle was increased and vibrissal (whisking) function was worsened. CONCLUSIONS: The use of 3-way-conduit provides no advantages to classic coaptation. Should the latter be impossible (too large interstump defects requiring too long interpositional nerve grafts), this type of reconstruction may be applied. (230 words).


Assuntos
Aorta Abdominal/transplante , Axônios , Nervo Facial/cirurgia , Regeneração Nervosa , Procedimentos Neurocirúrgicos , Procedimentos Cirúrgicos Reconstrutivos , Anastomose Cirúrgica , Animais , Axônios/patologia , Axônios/fisiologia , Músculos Faciais/inervação , Músculos Faciais/patologia , Nervo Facial/patologia , Nervo Facial/fisiopatologia , Traumatismos do Nervo Facial/cirurgia , Feminino , Nervo Hipoglosso/patologia , Nervo Hipoglosso/fisiopatologia , Nervo Hipoglosso/cirurgia , Atividade Motora , Regeneração Nervosa/fisiologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Ratos Wistar , Recuperação de Função Fisiológica , Vibrissas/inervação
11.
Theranostics ; 9(5): 1232-1246, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30867827

RESUMO

The study of human neuromuscular diseases has traditionally been performed in animal models, due to the difficulty of performing studies in human subjects. Despite the unquestioned value of animal models, inter-species differences hamper the translation of these findings to clinical trials. Tissue-engineered models of the neuromuscular junction (NMJ) allow for the recapitulation of the human physiology in tightly controlled in vitro settings. Methods: Here we report the first human patient-specific tissue-engineered model of the neuromuscular junction (NMJ) that combines stem cell technology with tissue engineering, optogenetics, microfabrication and image processing. The combination of custom-made hardware and software allows for repeated, quantitative measurements of NMJ function in a user-independent manner. Results: We demonstrate the utility of this model for basic and translational research by characterizing in real time the functional changes during physiological and pathological processes. Principal Conclusions: This system holds great potential for the study of neuromuscular diseases and drug screening, allowing for the extraction of quantitative functional data from a human, patient-specific system.


Assuntos
Modelos Teóricos , Doenças Neuromusculares/patologia , Doenças Neuromusculares/fisiopatologia , Optogenética/métodos , Engenharia Tecidual/métodos , Humanos , Células-Tronco Pluripotentes Induzidas/fisiologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiologia , Junção Neuromuscular/fisiopatologia
12.
Int J Mol Sci ; 20(6)2019 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-30917493

RESUMO

Despite the relevant research efforts, the causes of amyotrophic lateral sclerosis (ALS) are still unknown and no effective cure is available. Many authors suggest that ALS is a multi-system disease caused by a network failure instead of a cell-autonomous pathology restricted to motoneurons. Although motoneuronal loss is the critical hallmark of ALS given their specific vulnerability, other cell populations, including muscle and glial cells, are involved in disease onset and progression, but unraveling their specific role and crosstalk requires further investigation. In particular, little is known about the plastic changes of the degenerating motor system. These spontaneous compensatory processes are unable to halt the disease progression, but their elucidation and possible use as a therapeutic target represents an important aim of ALS research. Genetic animal models of disease represent useful tools to validate proven hypotheses or to test potential therapies, and the conception of novel hypotheses about ALS causes or the study of pathogenic mechanisms may be advantaged by the use of relatively simple in vivo models recapitulating specific aspects of the disease, thus avoiding the inclusion of too many confounding factors in an experimental setting. Here, we used a neurotoxic model of spinal motoneuron depletion induced by injection of cholera toxin-B saporin in the gastrocnemius muscle to investigate the possible occurrence of compensatory changes in both the muscle and spinal cord. The results showed that, following the lesion, the skeletal muscle became atrophic and displayed electromyographic activity similar to that observed in ALS patients. Moreover, the changes in muscle fiber morphology were different from that observed in ALS models, thus suggesting that some muscular effects of disease may be primary effects instead of being simply caused by denervation. Notably, we found plastic changes in the surviving motoneurons that can produce a functional restoration probably similar to the compensatory changes occurring in disease. These changes could be at least partially driven by glutamatergic signaling, and astrocytes contacting the surviving motoneurons may support this process.


Assuntos
Atrofia Muscular Espinal/fisiopatologia , Junção Neuromuscular/fisiopatologia , Plasticidade Neuronal , Animais , Toxina da Cólera/toxicidade , Masculino , Camundongos , Músculo Esquelético/efeitos dos fármacos , Músculo Esquelético/fisiopatologia , Atrofia Muscular Espinal/etiologia , Atrofia Muscular Espinal/patologia , Junção Neuromuscular/patologia , Saporinas/toxicidade , Medula Espinal/patologia , Medula Espinal/fisiopatologia
13.
Biochim Biophys Acta Mol Basis Dis ; 1865(6): 1579-1591, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30904609

RESUMO

Evolutionarily conserved homeostatic systems have been shown to modulate synaptic efficiency at the neuromuscular junctions of organisms. While advances have been made in identifying molecules that function presynaptically during homeostasis, limited information is currently available on how postsynaptic alterations affect presynaptic function. We previously identified a role for postsynaptic Dystrophin in the maintenance of evoked neurotransmitter release. We herein demonstrated that Dystrobrevin, a member of the Dystrophin Glycoprotein Complex, was delocalized from the postsynaptic region in the absence of Dystrophin. A newly-generated Dystrobrevin mutant showed elevated evoked neurotransmitter release, increased bouton numbers, and a readily releasable pool of synaptic vesicles without changes in the function or numbers of postsynaptic glutamate receptors. In addition, we provide evidence to show that the highly conserved Cdc42 Rho GTPase plays a key role in the postsynaptic Dystrophin/Dystrobrevin pathway for synaptic homeostasis. The present results give novel insights into the synaptic deficits underlying Duchenne Muscular Dystrophy affected by a dysfunctional Dystrophin Glycoprotein complex.


Assuntos
Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Proteínas Associadas à Distrofina/genética , Distrofina/genética , Junção Neuromuscular/genética , Proteína cdc42 de Ligação ao GTP/genética , Animais , Animais Geneticamente Modificados , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Distrofina/deficiência , Proteínas Associadas à Distrofina/metabolismo , Regulação da Expressão Gênica , Homeostase/genética , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Junção Neuromuscular/metabolismo , Junção Neuromuscular/patologia , Potenciais Sinápticos/genética , Transmissão Sináptica , Vesículas Sinápticas/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo
14.
Int J Exp Pathol ; 100(1): 49-59, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30773727

RESUMO

Knowledge of skeletal muscle adaptations is important to understand the functional deficits in cerebral palsy (CP). This study aimed to investigate the morphofunctional characteristics of skeletal muscle in a CP animal model. Initially, pregnant Wistar rats were injected intraperitoneally with saline or lipopolysaccharide over the last five days of pregnancy. The control group (n = 8) consisted of male pups born to females injected with saline. The CP group (n = 8) consisted of male pups born to females injected with lipopolysaccharide, which were submitted to perinatal anoxia [day of birth, postnatal day 0 (P0)] and sensorimotor restriction (P1-P30). The open-field test was undertaken on P29 and P45. On P48, the animals were weighed, and the plantaris muscle was collected and its weight and length were measured. Transverse sections were stained with haematoxylin-eosin, NADH-TR, Masson's trichrome and non-specific esterase reaction for analysis. and transmission electron microscopy was performed. In the CP group, reductions were observed in mobility time, number of crossings and rearing frequency, body weight, muscle weight and length, and nucleus-to-fibre and capillary-to-fibre ratios. There was a statistically significant increase in the percentage area of the muscle section occupied by collagen; reduction in the area and increase in the number of type I muscle fibres; increase in myofibrillar disorganization and Z-line disorganization and dissolution; and reduction in the area and largest and smallest diameters of neuromuscular junctions. Thus this animal model of CP produced morphofunctional alterations in skeletal muscle, that were associated with evidence of motor deficits as demonstrated by the open-field test.


Assuntos
Paralisia Cerebral/patologia , Paralisia Cerebral/fisiopatologia , Locomoção , Atividade Motora , Músculo Esquelético/fisiopatologia , Músculo Esquelético/ultraestrutura , Animais , Paralisia Cerebral/induzido quimicamente , Paralisia Cerebral/metabolismo , Colágeno/metabolismo , Modelos Animais de Doenças , Feminino , Hipóxia/complicações , Lipopolissacarídeos , Masculino , Músculo Esquelético/metabolismo , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Gravidez , Ratos Wistar
15.
Neuroscience ; 404: 91-101, 2019 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-30738855

RESUMO

Septins (Sept) are highly conserved Guanosine-5'-triphosphate (GTP)-binding cytoskeletal proteins involved in neuronal signaling in the central nervous system but their involvement in signal transmission in peripheral synapses remains unclear. Sept5 and Sept9 proteins were detected in mouse peripheral neuromuscular junctions by immunofluorescence with a greater degree of co-localization with presynaptic than postsynaptic membranes. Preincubation of neuromuscular junction preparations with the inhibitor of Sept dynamics, forchlorfenuron (FCF), decreased co-localization of Sept with presynaptic membranes. FCF introduced ex vivo or in vivo had no effect on the amplitude of the spontaneous endplate currents (EPCs), indicating the absence of postsynaptic effects of FCF. However, FCF decreased acetylcholine (ACh) quantal release in response to nerve stimulation, reduced the amplitude of evoked quantal currents and decreased the number of quanta with long synaptic delays, demonstrating the presynaptic action of FCF. Nevertheless, FCF had no effect on the amplitude of calcium transient in nerve terminals, as detected by calcium-sensitive dye, and slightly decreased the ratio of the second response amplitude to the first one in paired-pulse experiments. These results suggest that FCF-induced decrease in ACh quantal secretion is not due to a decrease in Ca2+ influx but is likely related to the impairment of later stages occurring after Ca2+ entry, such as trafficking, docking or membrane fusion of synaptic vesicles. Therefore, Sept9 and Sept5 are abundantly expressed in presynaptic membranes, and disruption of Sept dynamics suppresses the evoked synchronous and delayed asynchronous quantal release of ACh, strongly suggesting an important role of Sept in the regulation of neurotransmission in peripheral synapses.


Assuntos
Potencial Evocado Motor/fisiologia , Junção Neuromuscular/patologia , Septinas/fisiologia , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Diafragma/inervação , Diafragma/fisiologia , Camundongos , Camundongos Endogâmicos BALB C , Nervo Frênico/fisiologia
16.
Exp Neurol ; 315: 60-71, 2019 05.
Artigo em Inglês | MEDLINE | ID: mdl-30731076

RESUMO

Peripheral nerve regeneration following injury is often incomplete, resulting in significant personal and socioeconomic costs. Although a conditioning crush lesion prior to surgical nerve transection and repair greatly promotes nerve regeneration and functional recovery, feasibility and ethical considerations have hindered its clinical applicability. In a recent proof of principle study, we demonstrated that conditioning electrical stimulation (CES) had effects on early nerve regeneration, similar to that seen in conditioning crush lesions (CCL). To convincingly determine its clinical utility, establishing the effects of CES on target reinnervation and functional outcomes is of utmost importance. In this study, we found that CES improved nerve regeneration and reinnervation well beyond that of CCL. Specifically, compared to CCL, CES resulted in greater intraepidermal skin and NMJ reinnervation, and greater physiological and functional recovery including mechanosensation, compound muscle action potential on nerve conduction studies, normalization of gait pattern, and motor performance on the horizontal ladder test. These findings have direct clinical relevance as CES could be delivered at the bedside before scheduled nerve surgery.


Assuntos
Terapia por Estimulação Elétrica , Regeneração Nervosa , Potenciais de Ação , Animais , Marcha , Masculino , Compressão Nervosa , Condução Nervosa , Junção Neuromuscular/patologia , Traumatismos dos Nervos Periféricos/patologia , Desempenho Psicomotor , Ratos , Ratos Sprague-Dawley , Recuperação de Função Fisiológica , Sensação , Pele/inervação
17.
Toxicology ; 416: 62-74, 2019 03 15.
Artigo em Inglês | MEDLINE | ID: mdl-30682440

RESUMO

BACKGROUND: Carbamates physostigmine and pyridostigmine have been used as a pretreatment against poisoning with nerve agents in order to reversibly inhibit and thus protect from irreversible inhibition a portion of acetylcholinesterase (AChE) in brain and respiratory muscles that is crucial for survival. Memantine, an adamantine derivative, has emerged as a promising alternative to carbamates, since it prevented the fasciculations and skeletal muscle necrosis induced by carbamates and organophosphates, including nerve agents. AIM: This experimental study was undertaken in order to investigate and compare the protective and behavioural effects of memantine and standard carbamates physostigmine and pyridostigmine in rats poisoned with soman and treated with atropine, oxime HI-6 and diazepam. Another goal was to elucidate the mechanisms of the antidotal effect of memantine and its potential synergism with standard antidotes against nerve agents. MATERIALS AND METHODS: Male Wistar rats were used throughout the experiments. In dose-finding experiments memantine was administered at dose interval 0-72 mg/kg sc 60 min before sc injection of soman. In time-finding experiments memantine was injected 18 mg/kg sc 0-1440 min before soman. Standard treatment antidotes - atropine 10 mg/kg, HI-6 50 mg/kg and diazepam 2.5 mg/kg - were administered im within 15 s post-exposure. Soman 0.75 LD50 was used to study its inhibitions of neuromuscular transmission on the phrenic nerve-diaphragm preparation in situ and of tissue AChE activity. Behavioural effects of the prophylactic antidotes were investigated by means of the rotarod test. Based on these data therapeutic index and therapeutic width was calculated for all three prophylactic agents. RESULTS: Memantine pretreatment (18 mg/kg sc) produced in rats poisoned with soman significantly better protective ratios (PRs) than the two carbamates - 1.25 when administered alone and 2.3 when combined with atropine pretreatment and 6.33 and 7.23 with atropine/HI-6 and atropine/HI-6/diazepam post-exposure therapy, respectively. The highest PR of 10.11 obtained in Atr/HI-6-treated rats was achieved after pretreatment with memantine 36 mg/kg. This additional protection lasted for 8 h. All three prophylactic regimens antagonised the soman-induced neuromuscular blockade, but the effect of memantine was fastest. Pretreatment with memantine assured higher AChE activity in brain and diaphragm than in unpretreated rats (46% vs 28% and 68% vs. 38%, respectively). All three prophylactic regimens affected the rotarod performance in rats, but the effect of memantine was relatively strongest. Memantine and pyridostigmine had lowest and highest therapeutic index and therapeutic width, respectively. CONCLUSIONS: Although memantine assures better and longer-lasting protection against soman poisoning in rats than the two carbamates, its small therapeutic index and narrow therapeutic width seriously limit its potential as a pretreatment agent. Despite its behavioural effects, memantine seems to be beneficial antidote when administered after soman, along with atropine/HI-6/diazepam therapy. Mechanism of the antidotal effect of memantine against soman poisoning appears to be a combination of AChE-protecting and NMDA receptor-blocking action.


Assuntos
Antídotos/farmacologia , Substâncias para a Guerra Química , Inibidores da Colinesterase , Memantina/farmacologia , Junção Neuromuscular/efeitos dos fármacos , Intoxicação por Organofosfatos/prevenção & controle , Soman , Acetilcolinesterase/metabolismo , Animais , Atropina/farmacologia , Comportamento Animal/efeitos dos fármacos , Diazepam/farmacologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Quimioterapia Combinada , Proteínas Ligadas por GPI/metabolismo , Masculino , Junção Neuromuscular/enzimologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Intoxicação por Organofosfatos/enzimologia , Intoxicação por Organofosfatos/patologia , Intoxicação por Organofosfatos/fisiopatologia , Oximas/farmacologia , Compostos de Piridínio/farmacologia , Ratos Wistar , Receptores de N-Metil-D-Aspartato/metabolismo , Transmissão Sináptica/efeitos dos fármacos
18.
G3 (Bethesda) ; 9(1): 297-303, 2019 01 09.
Artigo em Inglês | MEDLINE | ID: mdl-30478082

RESUMO

The neuromuscular junction (NMJ) consists of three components, namely presynaptic motor neurons, postsynaptic muscle fibers and perisynaptic Schwann cells (PSCs). The role of Schwann cells (SCs) in regulating NMJ structural and functional development remains unclear. In this study, mice with conditional inactivation of neurofibromin 1 (Nf1) and phosphatase and tensin homolog (Pten), specifically in SCs, resulted in delayed NMJ maturation that led to delayed muscle growth, recapitulating the muscular dystrophy condition observed in human neurofibromatosis type I syndrome (NF1) patients. Expression levels of NMJ development related molecules such as cholinergic receptor, nicotinic, alpha polypeptide 1 (Chrna1), agrin (Agrn), dystrophin, muscular dystrophy (Dmd), laminin, beta 2 (Lamb2) and dystroglycan 1 (Dag1) were also downregulated. To further explore the molecular alterations in these SCs, NF1- and PTEN-related pathways were analyzed in mutant sciatic nerves. As expected, hyperactive RAS/PI3K/AKT/mTOR signaling pathways were identified, suggesting the importance of these pathways for NMJ development, and subsequent muscle maturation.


Assuntos
Desenvolvimento Muscular/genética , Neurofibromina 1/genética , Junção Neuromuscular/genética , PTEN Fosfo-Hidrolase/genética , Animais , Distroglicanas/genética , Distrofina/genética , Regulação da Expressão Gênica/genética , Humanos , Laminina/genética , Camundongos , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Músculo Esquelético/crescimento & desenvolvimento , Músculo Esquelético/patologia , Junção Neuromuscular/patologia , Receptores Nicotínicos/genética , Células de Schwann/metabolismo , Células de Schwann/patologia , Nervo Isquiático/metabolismo , Nervo Isquiático/patologia , Transdução de Sinais/genética , Sinapses/genética , Sinapses/patologia
19.
Muscle Nerve ; 59(1): 108-115, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30121966

RESUMO

INTRODUCTION: With a view to simplifying surgical techniques for selective laryngeal reinnervation, we addressed the question of whether it is feasible to receive additional innervation by a partially denervated muscle using an infrahyoid muscle model. METHODS: In 90 rats (6 groups of 15), phrenic nerve transfer was used to reinnervate the sternothyroid muscle. In some cases, residual innervation by the original nerve was present. Three months later we performed electromyographic studies, contraction strength measurements, histologic assessment, and retrograde labeling. RESULTS: Muscles reinnervated by the phrenic nerve had a greater "dual-response" rate (in terms of nerve latency, contraction strength, and retrograde labeling) than muscles in the control groups. DISCUSSION: The phrenic nerve can impart its inspiratory properties to an initially denervated strap muscle-even when residual innervation is present. The preservation of contractile potential confirmed the feasibility of dual innervation in a previously injured muscle. Muscle Nerve 59:108-115, 2019.


Assuntos
Denervação Muscular/métodos , Doenças Musculares/cirurgia , Transferência de Nervo/métodos , Nervo Frênico/fisiologia , Animais , Axônios/patologia , Modelos Animais de Doenças , Eletromiografia , Fluxo Expiratório Forçado , Placa Motora/fisiopatologia , Contração Muscular/fisiologia , Força Muscular/fisiologia , Doenças Musculares/etiologia , Músculos do Pescoço/fisiopatologia , Condução Nervosa/fisiologia , Junção Neuromuscular/patologia , Ratos , Ratos Wistar , Estatísticas não Paramétricas
20.
Neurobiol Dis ; 121: 148-162, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30290270

RESUMO

Mutations in the gene encoding the RNA-binding protein TDP-43 cause amyotrophic lateral sclerosis (ALS), clinically and pathologically indistinguishable from the majority of 'sporadic' cases of ALS, establishing altered TDP-43 function and distribution as a primary mechanism of neurodegeneration. Transgenic mouse models in which TDP-43 is overexpressed only partially recapitulate the key cellular pathology of human ALS, but may also lead to non-specific toxicity. To avoid the potentially confounding effects of overexpression, and to maintain regulated spatio-temporal and cell-specific expression, we generated mice in which an 80 kb genomic fragment containing the intact human TDP-43 locus (either TDP-43WT or TDP-43M337V) and its regulatory regions was integrated into the Rosa26 (Gt(ROSA26)Sor) locus in a single copy. At 3 months of age, TDP-43M337V mice are phenotypically normal but by around 6 months develop progressive motor function deficits associated with loss of neuromuscular junction integrity, leading to a reduced lifespan. RNA sequencing shows that widespread mis-splicing is absent prior to the development of a motor phenotype, though differential expression analysis reveals a distinct transcriptional profile in pre-symptomatic TDP-43M337V spinal cords. Despite the presence of clear motor abnormalities, there was no evidence of TDP-43 cytoplasmic aggregation in vivo at any timepoint. In primary embryonic spinal motor neurons and in embryonic stem cell (ESC)-derived motor neurons, mutant TDP-43 undergoes cytoplasmic mislocalisation, and is associated with altered stress granule assembly and dynamics. Overall, this mouse model provides evidence that ALS may arise through acquired TDP-43 toxicity associated with defective stress granule function. The normal phenotype until 6 months of age can facilitate the study of early pathways underlying ALS.


Assuntos
Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Proteínas de Ligação a DNA/genética , Neurônios Motores/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Expressão Gênica , Força da Mão , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/patologia , Mutação , Junção Neuromuscular/patologia , Proteínas de Ligação a RNA/metabolismo , Teste de Desempenho do Rota-Rod
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