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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.
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
3.
Muscle Nerve ; 60(6): 648-657, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31449669

RESUMO

Although myopathies and neuromuscular junction disorders are typically distinct, their coexistence has been reported in several inherited and acquired conditions. Affected individuals have variable clinical phenotypes but typically display both a decrement on repetitive nerve stimulation and myopathic findings on muscle biopsy. Inherited causes include myopathies related to mutations in BIN1, DES, DNM2, GMPPB, MTM1, or PLEC and congenital myasthenic syndromes due to mutations in ALG2, ALG14, COL13A1, DOK7, DPAGT1, or GFPT1. Additionally, a decrement due to muscle fiber inexcitability is observed in certain myotonic disorders. The identification of a defect of neuromuscular transmission in an inherited myopathy may assist in establishing a molecular diagnosis and in selecting patients who would benefit from pharmacological correction of this defect. Acquired cases meanwhile stem from the co-occurrence of myasthenia gravis or Lambert-Eaton myasthenic syndrome with an immune-mediated myopathy, which may be due to paraneoplastic disorders or exposure to immune checkpoint inhibitors.


Assuntos
Músculo Esquelético/fisiopatologia , Doenças Musculares/fisiopatologia , Síndromes Miastênicas Congênitas/fisiopatologia , Junção Neuromuscular/fisiopatologia , Cardiomiopatias/complicações , Cardiomiopatias/patologia , Cardiomiopatias/fisiopatologia , Eletrodiagnóstico , Eletromiografia , Humanos , Músculo Esquelético/patologia , Doenças Musculares/complicações , Doenças Musculares/patologia , Distrofias Musculares/complicações , Distrofias Musculares/patologia , Distrofias Musculares/fisiopatologia , Miastenia Gravis/complicações , Miastenia Gravis/patologia , Miastenia Gravis/fisiopatologia , Síndromes Miastênicas Congênitas/complicações , Síndromes Miastênicas Congênitas/patologia , Miopatias Congênitas Estruturais/complicações , Miopatias Congênitas Estruturais/patologia , Miopatias Congênitas Estruturais/fisiopatologia , Transtornos Miotônicos/complicações , Transtornos Miotônicos/patologia , Transtornos Miotônicos/fisiopatologia , Condução Nervosa
4.
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
5.
Handb Clin Neurol ; 160: 281-301, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31277854

RESUMO

An important component of needle EMG entails recording and interpreting the electrical signals generated from motor units during voluntary contraction. The recorded motor unit potentials (MUPs) reflect the number of motor units within a muscle and the distribution and density of muscle fibers within a motor unit within a portion of a muscle. Various MUP parameters are assessed to determine the integrity of the motor units, including recruitment, stability, phases and turns, duration, and amplitude. Each of these parameters is altered in a different way in various neuromuscular diseases. In neurogenic disorders, the earliest changes occur in the recruitment pattern of motor units followed by MUP morphologic changes (increased MUP phases and duration) as reinnervation occurs. MUP instability, indicating impaired neuromuscular transmission, also occurs in reinnervating neurogenic disorders as well as in neuromuscular junction disorders. In myopathies, a reduction in the size of the motor unit is manifested by MUPs of low amplitude and short duration. Interpreting the voluntary MUP changes along with spontaneous activity helps to determine the type, severity, and temporal course of neuromuscular diseases.


Assuntos
Eletromiografia/métodos , Doenças Neuromusculares/fisiopatologia , Junção Neuromuscular/fisiologia , Recrutamento Neurofisiológico/fisiologia , Humanos , Doenças Neuromusculares/diagnóstico , Junção Neuromuscular/fisiopatologia
6.
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
7.
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
8.
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
9.
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
10.
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
11.
Mar Biotechnol (NY) ; 21(1): 52-64, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30443836

RESUMO

The protein level of muscle-specific human NogoA is abnormally upregulated in amyotrophic lateral sclerosis (ALS) mice and patients. On the other hand, while the presence of miR-206 in muscle cells delays onset and death in ALS, the relationship between these two phenomena remains unclear. Mammalian NogoA protein, also known as Reticulon 4a (Rtn4a), plays an important role in inhibiting the outgrowth of motor neurons. Our group previously identified zebrafish rtn4al as the target gene of miR-206 and found that knockdown of miR-206 increases rtn4al mRNA and Rtn4al protein in zebrafish embryos. It can be concluded from these results that neurite outgrowth of motor neurons is inhibited by Rtn4a1, which is entirely consistent with overexpression of either human NogoA or zebrafish homolog Rtn4al. Since an animal model able to express NogoA/rtn4al at the mature stage is unavailable, we generated a zebrafish transgenic line, Tg(Zα:TetON-Rtn4al), which conditionally and specifically overexpresses Rtn4al in the muscle tissue. After doxycycline induction, adult zebrafish displayed denervation at neuromuscular junction during the first week, then muscle disintegration and split myofibers during the third week, and, finally, significant weight loss in the sixth week. These results suggest that this zebrafish transgenic line, representing the inducible overexpression of Rtn4a1 in muscle, may provide an alternative animal model with which to study ALS because it exhibits ALS-like phenotype.


Assuntos
Esclerose Amiotrófica Lateral/genética , Modelos Animais de Doenças , Neurônios Motores/metabolismo , Proteínas da Mielina/genética , Junção Neuromuscular/metabolismo , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Esclerose Amiotrófica Lateral/metabolismo , Esclerose Amiotrófica Lateral/fisiopatologia , Animais , Animais Geneticamente Modificados , Doxiciclina/farmacologia , Embrião não Mamífero , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Morfolinos/genética , Morfolinos/metabolismo , Neurônios Motores/patologia , Músculo Esquelético/metabolismo , Músculo Esquelético/patologia , Músculo Esquelético/fisiopatologia , Proteínas da Mielina/agonistas , Proteínas da Mielina/antagonistas & inibidores , Proteínas da Mielina/metabolismo , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Proteínas Nogo/agonistas , Proteínas Nogo/genética , Proteínas Nogo/metabolismo , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/metabolismo , Fenótipo , Plasmídeos/química , Plasmídeos/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/agonistas , Proteínas de Peixe-Zebra/antagonistas & inibidores , Proteínas de Peixe-Zebra/metabolismo
12.
Neurobiol Dis ; 124: 340-352, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30528255

RESUMO

Amyotrophic lateral sclerosis (ALS), a neurodegenerative disease affecting motor neurons, is characterized by rapid decline of motor function and ultimately respiratory failure. As motor neuron death occurs late in the disease, therapeutics that prevent the initial disassembly of the neuromuscular junction may offer optimal functional benefit and delay disease progression. To test this hypothesis, we treated the SOD1G93A mouse model of ALS with an agonist antibody to muscle specific kinase (MuSK), a receptor tyrosine kinase required for the formation and maintenance of the neuromuscular junction. Chronic MuSK antibody treatment fully preserved innervation of the neuromuscular junction when compared with control-treated mice; however, no preservation of diaphragm function, motor neurons, or survival benefit was detected. These data show that anatomical preservation of neuromuscular junctions in the diaphragm via MuSK activation does not correlate with functional benefit in SOD1G93A mice, suggesting caution in employing MuSK activation as a therapeutic strategy for ALS patients.


Assuntos
Esclerose Amiotrófica Lateral/enzimologia , Esclerose Amiotrófica Lateral/fisiopatologia , Diafragma/fisiopatologia , Junção Neuromuscular/fisiopatologia , Receptores Proteína Tirosina Quinases/agonistas , Esclerose Amiotrófica Lateral/patologia , Animais , Diafragma/patologia , Modelos Animais de Doenças , Ativação Enzimática/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios Motores/patologia , Junção Neuromuscular/patologia , Superóxido Dismutase-1/genética
13.
Gut ; 68(7): 1210-1223, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-30228216

RESUMO

OBJECTIVE: To determine if human colonic neuromuscular functions decline with increasing age. DESIGN: Looking for non-specific changes in neuromuscular function, a standard burst of electrical field stimulation (EFS) was used to evoke neuronally mediated (cholinergic/nitrergic) contractions/relaxations in ex vivomuscle strips of human ascending and descending colon, aged 35-91 years (macroscopically normal tissue; 239 patients undergoing cancer resection). Then, to understand mechanisms of change, numbers and phenotype of myenteric neurons (30 306 neurons stained with different markers), densities of intramuscular nerve fibres (51 patients in total) and pathways involved in functional changes were systematically investigated (by immunohistochemistry and use of pharmacological tools) in elderly (≥70 years) and adult (35-60 years) groups. RESULTS: With increasing age, EFS was more likely to evoke muscle relaxation in ascending colon instead of contraction (linear regression: n=109, slope 0.49%±0.21%/year, 95% CI), generally uninfluenced by comorbidity or use of medications. Similar changes were absent in descending colon. In the elderly, overall numbers of myenteric and neuronal nitric oxide synthase-immunoreactive neurons and intramuscular nerve densities were unchanged in ascending and descending colon, compared with adults. In elderly ascending, not descending, colon numbers of cell bodies exhibiting choline acetyltransferase immunoreactivity increased compared with adults (5.0±0.6 vs 2.4±0.3 neurons/mm myenteric plexus, p=0.04). Cholinergically mediated contractions were smaller in elderly ascending colon compared with adults (2.1±0.4 and 4.1±1.1 g-tension/g-tissue during EFS; n=25/14; p=0.04); there were no changes in nitrergic function or in ability of the muscle to contract/relax. Similar changes were absent in descending colon. CONCLUSION: In ascending not descending colon, ageing impairs cholinergic function.


Assuntos
Colo Ascendente/patologia , Colo Ascendente/fisiopatologia , Colo Descendente/patologia , Colo Descendente/fisiopatologia , Contração Muscular/fisiologia , Fibras Nervosas/patologia , Adulto , Fatores Etários , Idoso , Idoso de 80 Anos ou mais , Colo Ascendente/inervação , Colo Descendente/inervação , Estimulação Elétrica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fibras Nervosas/fisiologia , Vias Neurais/patologia , Vias Neurais/fisiopatologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Técnicas de Cultura de Tecidos
14.
Biogerontology ; 20(2): 213-223, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30560324

RESUMO

It is known that both exercise and aging influence neuromuscular performance; however their effects on post-exercise recovery are largely unknown. To examine how exercise training and aging might affect post-exercise recovery, the function of muscles taken from young, and aged male rats assigned to exercise, or control conditions was assessed with ex vivo procedures using indirect (nerve endings), and direct (sarcolemma) stimulation at different times (Initial, Final min of, and Recovery i.e. 1 min post, from 5 min of stimulation). Results revealed that initially, strength of indirectly stimulated young, male muscles was significantly (P = 0.05) greater than aged ones, but after continuous stimulation, aged and young muscles displayed similar strength, and controls showed more strength than trained muscles (P = 0.02). All groups except young controls exhibited significant recovery with 1 min of rest (P = 0.03). Compared to indirect stimulation, direct stimulation resulted in greater peak tension at each time point examined (P < 0.05); young muscles again were stronger than aged ones initially (P = 0.003), but not by the conclusion of stimulation (P = 0.20). One min following the direct stimulation protocol, no significant recovery was observed by any of the four treatment groups. These data indicate that motor neurons limit neuromuscular function, and that the effects of fatigue are more severe during stimulation of young, compared to aged muscle. Finally, results presented here indicate that age and training status do interact to influence post-exertional recovery, at least among male neuromuscular systems.


Assuntos
Envelhecimento/fisiologia , Músculo Esquelético , Junção Neuromuscular , Condicionamento Físico Animal/fisiologia , Animais , Masculino , Músculo Esquelético/fisiologia , Músculo Esquelético/fisiopatologia , Junção Neuromuscular/fisiologia , Junção Neuromuscular/fisiopatologia , Ratos , Recuperação de Função Fisiológica/fisiologia
15.
Biosystems ; 176: 13-16, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30578825

RESUMO

The electrical activity of external anal sphincter can be registered with surface electromyography. This signals are known to be highly complex and nonlinear. This work aims in characterisation of the information carried in the signals by harvesting the concept of information entropy. We will focus of two classical measures of the complexity. Firstly the Shannon entropy is addressed. It is related to the probability spectrum of the possible states. Secondly the Spectral entropy is described, as a simple frequency-domain analog of the time-domain Shannon characteristics. We discuss the power spectra for separate time scales and present the characteristics which can represent the dynamics of electrical activity of this specific muscle group. We find that the rest and maximum contraction states represent rather different spectral characteristic of entropy, with close-to-normal contraction and negatively skewed rest state.


Assuntos
Eletromiografia/métodos , Entropia , Músculo Esquelético/fisiopatologia , Junção Neuromuscular/fisiopatologia , Neoplasias Retais/fisiopatologia , Idoso , Algoritmos , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neoplasias Retais/cirurgia , Processamento de Sinais Assistido por Computador
16.
PLoS Genet ; 14(12): e1007845, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30543681

RESUMO

Nucleoporins build the nuclear pore complex (NPC), which, as sole gate for nuclear-cytoplasmic exchange, is of outmost importance for normal cell function. Defects in the process of nucleocytoplasmic transport or in its machinery have been frequently described in human diseases, such as cancer and neurodegenerative disorders, but only in a few cases of developmental disorders. Here we report biallelic mutations in the nucleoporin NUP88 as a novel cause of lethal fetal akinesia deformation sequence (FADS) in two families. FADS comprises a spectrum of clinically and genetically heterogeneous disorders with congenital malformations related to impaired fetal movement. We show that genetic disruption of nup88 in zebrafish results in pleiotropic developmental defects reminiscent of those seen in affected human fetuses, including locomotor defects as well as defects at neuromuscular junctions. Phenotypic alterations become visible at distinct developmental stages, both in affected human fetuses and in zebrafish, whereas early stages of development are apparently normal. The zebrafish phenotypes caused by nup88 deficiency are rescued by expressing wild-type Nup88 but not the disease-linked mutant forms of Nup88. Furthermore, using human and mouse cell lines as well as immunohistochemistry on fetal muscle tissue, we demonstrate that NUP88 depletion affects rapsyn, a key regulator of the muscle nicotinic acetylcholine receptor at the neuromuscular junction. Together, our studies provide the first characterization of NUP88 in vertebrate development, expand our understanding of the molecular events causing FADS, and suggest that variants in NUP88 should be investigated in cases of FADS.


Assuntos
Artrogripose/genética , Genes Letais , Mutação , Complexo de Proteínas Formadoras de Poros Nucleares/genética , Alelos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Artrogripose/embriologia , Artrogripose/fisiopatologia , Consanguinidade , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Modelos Moleculares , Proteínas Musculares/metabolismo , Junção Neuromuscular/fisiopatologia , Complexo de Proteínas Formadoras de Poros Nucleares/química , Complexo de Proteínas Formadoras de Poros Nucleares/deficiência , Linhagem , Gravidez , Conformação Proteica , Receptores Nicotínicos/metabolismo , Homologia de Sequência de Aminoácidos , Peixe-Zebra/anormalidades , Peixe-Zebra/genética , Peixe-Zebra/fisiologia , Proteínas de Peixe-Zebra/deficiência , Proteínas de Peixe-Zebra/genética
17.
Dis Model Mech ; 11(11)2018 11 20.
Artigo em Inglês | MEDLINE | ID: mdl-30459155

RESUMO

Mucopolysaccharidosis VII (MPS VII) is a recessively inherited lysosomal storage disorder caused by ß-glucuronidase enzyme deficiency. The disease is characterized by widespread accumulation of non-degraded or partially degraded glycosaminoglycans, leading to cellular and multiple tissue dysfunctions. The patients exhibit diverse clinical symptoms, and eventually succumb to premature death. The only possible remedy is the recently approved enzyme replacement therapy, which is an expensive, invasive and lifelong treatment procedure. Small-molecule therapeutics for MPS VII have so far remained elusive primarily due to lack of molecular insights into the disease pathogenesis and unavailability of a suitable animal model that can be used for rapid drug screening. To address these issues, we developed a Drosophila model of MPS VII by knocking out the CG2135 gene, the fly ß-glucuronidase orthologue. The CG2135 -/- fly recapitulated cardinal features of MPS VII, such as reduced lifespan, progressive motor impairment and neuropathological abnormalities. Loss of dopaminergic neurons and muscle degeneration due to extensive apoptosis was implicated as the basis of locomotor deficit in this fly. Such hitherto unknown mechanistic links have considerably advanced our understanding of the MPS VII pathophysiology and warrant leveraging this genetically tractable model for deeper enquiry about the disease progression. We were also prompted to test whether phenotypic abnormalities in the CG2135 -/- fly can be attenuated by resveratrol, a natural polyphenol with potential health benefits. Indeed, resveratrol treatment significantly ameliorated neuromuscular pathology and restored normal motor function in the CG2135 -/- fly. This intriguing finding merits further preclinical studies for developing an alternative therapy for MPS VII.This article has an associated First Person interview with the first author of the paper.


Assuntos
Drosophila melanogaster/metabolismo , Atividade Motora , Mucopolissacaridose VII/tratamento farmacológico , Mucopolissacaridose VII/fisiopatologia , Junção Neuromuscular/patologia , Junção Neuromuscular/fisiopatologia , Resveratrol/uso terapêutico , Sequência de Aminoácidos , Animais , Modelos Animais de Doenças , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/efeitos dos fármacos , Marcação de Genes , Glucuronidase/química , Glucuronidase/metabolismo , Humanos , Atividade Motora/efeitos dos fármacos , Junção Neuromuscular/efeitos dos fármacos , Junção Neuromuscular/metabolismo , Fenótipo , Resveratrol/farmacologia
18.
Eur Neurol ; 80(3-4): 151-156, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30463071

RESUMO

INTRODUCTION: To illuminate the mechanism of neuromuscular junction involvement by analyzing the features of a slow-rate repetitive nerve simulation (RNS) and EMG in amyotrophic lateral sclerosis (ALS) patients. METHODS: We retrospectively analyzed relationship between clinical features and the decremental response in RNS in 184 sporadic ALS patients. RESULTS: (1) 44.3 and 43.5% of compound muscle action potentials (CMAP) decrement were more than -10% in deltoid muscle (Del) and Trap; (2) ALS patients were divided into RNS positive (RNS+) and RNS negative (RNS-) group according to decremental percentage of RNS greater or less than -10%. The diagnostic delay time was shorter and ALSFRS-r score was lower in RNS+ than RNS- group (p < 0.05), progression rate had no difference. The incidence of RNS+ decrement was higher in cervical enlargement onset and definite diagnostic degrees (p < 0.05); (3) In EMG+ group, the CMAP amplitude was lower (Axillary: 3.1 ± 1.91 vs. 5.92 ± 2.896, p = 0.000; Accessory: 2.68 ± 1.349 vs. 3.65 ± 1.53, p = 0.002), decremental percentage of RNS was higher (Axillary: -10.85 ± 7.508 vs. -5.43 ± 8.425, p = 0.000; Accessory: -13.11 ± 7.539 vs. -8.03 ± 5.999, p = 0.000) compared with needle EMG- group whether in Del or Trap; (4) Decremental response of RNS was positively correlated with the CMAP amplitude in Axillary and Accessory nerves (R = 0.201, p < 0.0001; R = 0.103, p < 0.0001). CONCLUSIONS: Our clinical results support the mechanism of decremental phenomenon of RNS is immature sprouts and unstable conduction by the degenerating axons in ALS patients. The more serious the axon damage, the more significant the RNS decremental response. But decremental response dose not effect disease progression.


Assuntos
Potenciais de Ação/fisiologia , Esclerose Amiotrófica Lateral/fisiopatologia , Junção Neuromuscular/fisiopatologia , Adulto , Idoso , Esclerose Amiotrófica Lateral/diagnóstico , Progressão da Doença , Estimulação Elétrica , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos
19.
Elife ; 72018 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-30320556

RESUMO

Despite being an early event in ALS, it remains unclear whether the denervation of neuromuscular junctions (NMJ) is simply the first manifestation of a globally degenerating motor neuron. Using in vivo imaging of single axons and their NMJs over a three-month period, we identify that single motor-units are dismantled asynchronously in SOD1G37R mice. We reveal that weeks prior to complete axonal degeneration, the dismantling of axonal branches is accompanied by contemporaneous new axonal sprouting resulting in synapse formation onto nearby NMJs. Denervation events tend to propagate from the first lost NMJ, consistent with a contribution of neuromuscular factors extrinsic to motor neurons, with distal branches being more susceptible. These results show that NMJ denervation in ALS is a complex and dynamic process of continuous denervation and new innervation rather than a manifestation of sudden global motor neuron degeneration.


Assuntos
Esclerose Amiotrófica Lateral/fisiopatologia , Neurônios Motores/patologia , Junção Neuromuscular/fisiopatologia , Superóxido Dismutase/metabolismo , Animais , Axônios/patologia , Modelos Animais de Doenças , Progressão da Doença , Camundongos Transgênicos , Modelos Biológicos , Mutação/genética
20.
JBI Database System Rev Implement Rep ; 16(10): 1922-1928, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30335038
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