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1.
Elife ; 82019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31868582

RESUMO

Animals generate diverse motor behaviors, yet how the same motor neurons (MNs) generate two distinct or antagonistic behaviors remains an open question. Here, we characterize Drosophila larval muscle activity patterns and premotor/motor circuits to understand how they generate forward and backward locomotion. We show that all body wall MNs are activated during both behaviors, but a subset of MNs change recruitment timing for each behavior. We used TEM to reconstruct a full segment of all 60 MNs and 236 premotor neurons (PMNs), including differentially-recruited MNs. Analysis of this comprehensive connectome identified PMN-MN 'labeled line' connectivity; PMN-MN combinatorial connectivity; asymmetric neuronal morphology; and PMN-MN circuit motifs that could all contribute to generating distinct behaviors. We generated a recurrent network model that reproduced the observed behaviors, and used functional optogenetics to validate selected model predictions. This PMN-MN connectome will provide a foundation for analyzing the full suite of larval behaviors.


Assuntos
Drosophila/fisiologia , Locomoção/fisiologia , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Animais , Análise por Conglomerados , Conectoma , Drosophila/embriologia , Drosophila melanogaster/fisiologia , Larva/fisiologia , Modelos Animais , Músculos/citologia , Músculos/diagnóstico por imagem , Músculos/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Optogenética , Sinapses/fisiologia , Sinapses/ultraestrutura
2.
Artif Cells Nanomed Biotechnol ; 47(1): 4240-4247, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31713439

RESUMO

Exploring the spatial relationship of various neuron pools in the spinal cord is crucial and difficult due to its complexity. The single-labelling tracing and sectioning were employed in previous studies exploring the distribution of spinal motor neuron pools, which could only delineate one single motor neuron pool in one specimen and could not achieve intact-tissue observation. Here, with combination of neuroanatomy tracing techniques and the optical clearing technique, we developed a multiple retrograde tracing method compatible with 3DISCO clearing. Fluoro-Gold, Fluoro-Ruby, Cholera Toxin Subunit B, Alexa Fluor 488 and 647 Conjugate were injected intramuscularly in hindlimbs of C57BL/6 adults. After labelling, the harvested spinal cords were optically cleared by 3DISCO method and imaged using confocal microscope. There were positive signals of all four tracers and four motor neurons pools targeting injected muscles were labelled. Three-dimension model of four motor neuron pools was successfully reconstructed based on tomography images showing the spatial relationship of different neuron pools. In conclusion, using this method, we first delineated the spatial relationship of four different motor neuron pools targeting four skeletal muscles in one spinal cord at the same time, which provide a holistic view of motor neuron pools in the spinal cord.


Assuntos
Microscopia/métodos , Fenômenos Ópticos , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios Motores/citologia , Traçadores Radioativos , Medula Espinal/citologia , Medula Espinal/diagnóstico por imagem
3.
Plast Reconstr Surg ; 144(6): 1359-1368, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31764653

RESUMO

BACKGROUND: The topographic distribution through histologic analysis of motor and sensory axons within peripheral nerves at the brachial plexus level is not clearly defined, as there has previously been little need to appreciate this microanatomy. A desire to better understand the topography of fascicle groups developed with the introduction of targeted muscle reinnervation. METHODS: Fourteen bilateral brachial plexus specimens from seven fresh human cadavers were harvested at the time of organ donation, and immunofluorescent staining of motor and sensory nerves with choline acetyltransferase and Neurofilament 200 was performed to determine whether a consistent somatotopic orientation exists at the brachial plexus level. RESULTS: There was significant variability in the number of fascicles at the level of the brachial plexus. Qualitative analysis of choline acetyltransferase staining demonstrated that although motor axons tended to be grouped in clusters, there were high degrees of variability in somatotopic orientation across specimens. The radial nerve demonstrated the highest number of total myelinated axons, whereas the median nerve exhibited the greatest number of motor axons. The ulnar nerve contained only 13 percent motor axons, which was significantly lower than the median, radial, and musculocutaneous nerves. CONCLUSIONS: There was no consistent somatotopic organization of motor and sensory axons of the mixed major nerves of the arm just distal to the brachial plexus, but clustering of motor axons may facilitate the splitting of nerves into primarily "motor" and "sensory" fascicles.


Assuntos
Axônios , Plexo Braquial/anatomia & histologia , Neurônios Motores/citologia , Idoso , Cadáver , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fibras Nervosas Mielinizadas , Células Receptoras Sensoriais/citologia
4.
Brain Nerve ; 71(11): 1279-1288, 2019 Nov.
Artigo em Japonês | MEDLINE | ID: mdl-31722314

RESUMO

Our laboratory previously established spinal motor neurons (MN) from induced-pluripotent stem cells (iPSCs) prepared from both sporadic and familial ALS patients, and successfully recapitulated disease-specific pathophysiological processes. We next searched for effective drugs capable of slowing the progression of ALS using a drug library of 1232 existing compounds and discovered that ropinirole hydrochloride prevented MN death. In December 2018, we started an investigator-initiated clinical trial testing ropinirole hydrochloride extended-release tablets in ALS patients. This is an on-going phase I/IIa randomized, double-blind, placebo-controlled, single-center, open-label continuation clinical trial (UMIN000034954). The primary aim is to assess the safety and tolerability of ropinirole hydrochloride in patients with ALS. We will also perform an efficacy evaluation using patient-derived iPSCs/MN. Major inclusion criteria were as follows: 1) 'clinically possible and laboratory-supported ALS', 'clinically probable ALS' or 'clinically definite ALS', according to the criteria for the diagnosis of ALS (El Escorial revised) and within 60 months after disease onset; 2) change in ALSFRS-R score of -2 to -5 points during the 12-week run-in period. Finally, 15 patients will be assigned to the active drug and 5 patients to the placebo. Our trial will be a touchstone trial for iPSC-based drug development strategies.


Assuntos
Esclerose Amiotrófica Lateral/tratamento farmacológico , Indóis/uso terapêutico , Células-Tronco Pluripotentes Induzidas , Neurônios Motores/citologia , Método Duplo-Cego , Humanos
5.
PLoS Biol ; 17(10): e3000480, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31613896

RESUMO

Many species execute ballistic escape reactions to avoid imminent danger. Despite fast reaction times, responses are often highly regulated, reflecting a trade-off between costly motor actions and perceived threat level. However, how sensory cues are integrated within premotor escape circuits remains poorly understood. Here, we show that in zebrafish, less precipitous threats elicit a delayed escape, characterized by flexible trajectories, which are driven by a cluster of 38 prepontine neurons that are completely separate from the fast escape pathway. Whereas neurons that initiate rapid escapes receive direct auditory input and drive motor neurons, input and output pathways for delayed escapes are indirect, facilitating integration of cross-modal sensory information. These results show that rapid decision-making in the escape system is enabled by parallel pathways for ballistic responses and flexible delayed actions and defines a neuronal substrate for hierarchical choice in the vertebrate nervous system.


Assuntos
Reação de Fuga/fisiologia , Córtex Motor/fisiologia , Neurônios Motores/fisiologia , Reconhecimento Fisiológico de Modelo/fisiologia , Ponte/fisiologia , Peixe-Zebra/fisiologia , Animais , Tomada de Decisões/fisiologia , Larva/fisiologia , Córtex Motor/citologia , Neurônios Motores/citologia , Ponte/citologia , Tempo de Reação/fisiologia
6.
Elife ; 82019 10 04.
Artigo em Inglês | MEDLINE | ID: mdl-31584430

RESUMO

Dendritic spines are specialized postsynaptic structures that transduce presynaptic signals, are regulated by neural activity and correlated with learning and memory. Most studies of spine function have focused on the mammalian nervous system. However, spine-like protrusions have been reported in C. elegans (Philbrook et al., 2018), suggesting that the experimental advantages of smaller model organisms could be exploited to study the biology of dendritic spines. Here, we used super-resolution microscopy, electron microscopy, live-cell imaging and genetics to show that C. elegans motor neurons have functional dendritic spines that: (1) are structurally defined by a dynamic actin cytoskeleton; (2) appose presynaptic dense projections; (3) localize ER and ribosomes; (4) display calcium transients triggered by presynaptic activity and propagated by internal Ca++ stores; (5) respond to activity-dependent signals that regulate spine density. These studies provide a solid foundation for a new experimental paradigm that exploits the power of C. elegans genetics and live-cell imaging for fundamental studies of dendritic spine morphogenesis and function.


Assuntos
Caenorhabditis elegans/citologia , Espinhas Dendríticas/ultraestrutura , Neurônios Motores/citologia , Animais , Microscopia Intravital , Microscopia Eletrônica , Microscopia de Fluorescência , Organelas/ultraestrutura
7.
PLoS One ; 14(10): e0223443, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31584985

RESUMO

Polyethylene glycol repair (PEG-fusion) of severed sciatic axons restores their axoplasmic and membrane continuity, prevents Wallerian degeneration, maintains muscle fiber innervation, and greatly improves recovery of voluntary behaviors. We examined alterations in spinal connectivity and motoneuron dendritic morphology as one potential mechanism for improved behavioral function after PEG-fusion. At 2-112 days after a single-cut or allograft PEG-fusion repair of transected or ablated sciatic nerves, the number, size, location, and morphology of motoneurons projecting to the tibialis anterior muscle were assessed by retrograde labeling. For both lesion types, labeled motoneurons were found in the appropriate original spinal segment, but also in inappropriate segments, indicating mis-pairings of proximal-distal segments of PEG-fused motor axons. Although the number and somal size of motoneurons was unaffected, dendritic distributions were altered, indicating that PEG-fusion preserves spinal motoneurons but reorganizes their connectivity. This spinal reorganization may contribute to the remarkable behavioral recovery seen after PEG-fusion repair.


Assuntos
Neurônios Motores/metabolismo , Condução Nervosa , Neuropatia Ciática/etiologia , Neuropatia Ciática/metabolismo , Potenciais de Ação , Aloenxertos , Animais , Contagem de Células , Dendritos/metabolismo , Modelos Animais de Doenças , Fenômenos Eletrofisiológicos , Feminino , Imuno-Histoquímica , Neurônios Motores/citologia , Regeneração Nervosa , Polietilenoglicóis , Ratos , Recuperação de Função Fisiológica , Neuropatia Ciática/reabilitação
8.
Elife ; 82019 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-31612855

RESUMO

Prior expectations of movement instructions can promote preliminary action planning and influence choices. We investigated how action priors affect action-goal encoding in premotor and parietal cortices and if they bias subsequent free choice. Monkeys planned reaches according to visual cues that indicated relative probabilities of two possible goals. On instructed trials, the reach goal was determined by a secondary cue respecting these probabilities. On rarely interspersed free-choice trials without instruction, both goals offered equal reward. Action priors induced graded free-choice biases and graded frontoparietal motor-goal activity, complementarily in two subclasses of neurons. Down-regulating neurons co-encoded both possible goals and decreased opposite-to-preferred responses with decreasing prior, possibly supporting a process of choice by elimination. Up-regulating neurons showed increased preferred-direction responses with increasing prior, likely supporting a process of computing net likelihood. Action-selection signals emerged earliest in down-regulating neurons of premotor cortex, arguing for an initiation of selection in the frontal lobe.


Assuntos
Antecipação Psicológica , Comportamento de Escolha/fisiologia , Tomada de Decisões/fisiologia , Macaca mulatta/fisiologia , Córtex Motor/fisiologia , Lobo Parietal/fisiologia , Potenciais de Ação/fisiologia , Animais , Sinais (Psicologia) , Eletrodos Implantados , Funções Verossimilhança , Macaca mulatta/anatomia & histologia , Macaca mulatta/psicologia , Córtex Motor/anatomia & histologia , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Movimento/fisiologia , Lobo Parietal/anatomia & histologia , Reconhecimento Visual de Modelos/fisiologia , Desempenho Psicomotor , Recompensa , Técnicas Estereotáxicas
9.
Nat Commun ; 10(1): 4197, 2019 09 13.
Artigo em Inglês | MEDLINE | ID: mdl-31519892

RESUMO

In all vertebrates, excitatory spinal interneurons execute dynamic adjustments in the timing and amplitude of locomotor movements. Currently, it is unclear whether interneurons responsible for timing control are distinct from those involved in amplitude control. Here, we show that in larval zebrafish, molecularly, morphologically and electrophysiologically distinct types of V2a neurons exhibit complementary patterns of connectivity. Stronger higher-order connections from type I neurons to other excitatory V2a and inhibitory V0d interneurons provide timing control, while stronger last-order connections from type II neurons to motor neurons provide amplitude control. Thus, timing and amplitude are coordinated by distinct interneurons distinguished not by their occupation of hierarchically-arranged anatomical layers, but rather by differences in the reliability and probability of higher-order and last-order connections that ultimately form a single anatomical layer. These findings contribute to our understanding of the origins of timing and amplitude control in the spinal cord.


Assuntos
Interneurônios/metabolismo , Locomoção/fisiologia , Animais , Interneurônios/citologia , Neurônios Motores/citologia , Neurônios Motores/metabolismo , Medula Espinal/citologia , Medula Espinal/metabolismo , Peixe-Zebra
10.
Elife ; 82019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31502540

RESUMO

How circuits assemble starting from stem cells is a fundamental question in developmental neurobiology. We test the hypothesis that, in neuronal stem cells, temporal transcription factors predictably control neuronal terminal features and circuit assembly. Using the Drosophila motor system, we manipulate expression of the classic temporal transcription factor Hunchback (Hb) specifically in the NB7-1 stem cell, which produces U motor neurons (MNs), and then we monitor dendrite morphology and neuromuscular synaptic partnerships. We find that prolonged expression of Hb leads to transient specification of U MN identity, and that embryonic molecular markers do not accurately predict U MN terminal features. Nonetheless, our data show Hb acts as a potent regulator of neuromuscular wiring decisions. These data introduce important refinements to current models, show that molecular information acts early in neurogenesis as a switch to control motor circuit wiring, and provide novel insight into the relationship between stem cell and circuit.


Assuntos
Proteínas de Ligação a DNA/biossíntese , Proteínas de Drosophila/biossíntese , Expressão Gênica , Neurônios Motores/fisiologia , Vias Neurais/embriologia , Junção Neuromuscular/fisiologia , Células-Tronco/fisiologia , Fatores de Transcrição/biossíntese , Animais , Drosophila , Neurônios Motores/citologia , Junção Neuromuscular/citologia , Células-Tronco/citologia
11.
Nat Commun ; 10(1): 3827, 2019 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-31444357

RESUMO

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown etiology. Although defects in nucleocytoplasmic transport (NCT) may be central to the pathogenesis of ALS and other neurodegenerative diseases, the molecular mechanisms modulating the nuclear pore function are still largely unknown. Here we show that genetic and pharmacological modulation of actin polymerization disrupts nuclear pore integrity, nuclear import, and downstream pathways such as mRNA post-transcriptional regulation. Importantly, we demonstrate that modulation of actin homeostasis can rescue nuclear pore instability and dysfunction caused by mutant PFN1 as well as by C9ORF72 repeat expansion, the most common mutation in ALS patients. Collectively, our data link NCT defects to ALS-associated cellular pathology and propose the regulation of actin homeostasis as a novel therapeutic strategy for ALS and other neurodegenerative diseases.


Assuntos
Actinas/metabolismo , Esclerose Amiotrófica Lateral/patologia , Neurônios Motores/patologia , Poro Nuclear/patologia , Profilinas/metabolismo , Acrilamidas/farmacologia , Actinas/ultraestrutura , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Esclerose Amiotrófica Lateral/genética , Biópsia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Linhagem Celular , Córtex Cerebral/citologia , Córtex Cerebral/patologia , Embrião de Mamíferos , Fibroblastos , Humanos , Microscopia Eletrônica de Transmissão , Neurônios Motores/citologia , Mutação , Poro Nuclear/efeitos dos fármacos , Poro Nuclear/ultraestrutura , Cultura Primária de Células , Profilinas/genética , Multimerização Proteica/efeitos dos fármacos , Multimerização Proteica/genética , Pele/citologia , Pele/patologia , Tiazóis/farmacologia , Tiazolidinas/farmacologia
12.
Brain Struct Funct ; 224(8): 2717-2731, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31375981

RESUMO

Medial rectus motoneurons are innervated by two main pontine inputs. The specific function of each of these two inputs remains to be fully understood. Indeed, selective partial deafferentation of medial rectus motoneurons, performed by the lesion of either the vestibular or the abducens input, initially induces similar changes in motoneuronal discharge. However, at longer time periods, the responses to both lesions are dissimilar. Alterations on eye movements and motoneuronal discharge induced by vestibular input transection recover completely 2 months post-lesion, whereas changes induced by abducens internuclear lesion are more drastic and permanent. Functional recovery could be due to some kind of plastic process, such as reactive synaptogenesis, developed by the remaining intact input, which would occupy the vacant synaptic spaces left after lesion. Herein, by means of confocal microscopy, immunocytochemistry and retrograde labeling, we attempt to elucidate the possible plastic processes that take place after partial deafferentation of medial rectus motoneuron. 48 h post-injury, both vestibular and abducens internuclear lesions produced a reduced synaptic coverage on these motoneurons. However, 96 h after vestibular lesion, there was a partial recovery in the number of synaptic contacts. This suggests that there was reactive synaptogenesis. This recovery was preceded by an increase in somatic neurotrophin content, suggesting a role of these molecules in presynaptic axonal sprouting. The rise in synaptic coverage might be due to terminal sprouting performed by the remaining main input, i.e., abducens internuclear neurons. The present results may improve the understanding of this apparently redundant input system.


Assuntos
Neurônios Motores/fisiologia , Plasticidade Neuronal , Músculos Oculomotores/fisiologia , Animais , Denervação/métodos , Movimentos Oculares , Masculino , Neurônios Motores/citologia , Vias Neurais/fisiologia , Músculos Oculomotores/inervação , Ratos Wistar
13.
Nat Commun ; 10(1): 3784, 2019 08 22.
Artigo em Inglês | MEDLINE | ID: mdl-31439839

RESUMO

Disruption in membrane excitability contributes to malfunction and differential vulnerability of specific neuronal subpopulations in a number of neurological diseases. The adaptor protein p11, and background potassium channel TASK1, have overlapping distributions in the CNS. Here, we report that the transcription factor Sp1 controls p11 expression, which impacts on excitability by hampering functional expression of TASK1. In the SOD1-G93A mouse model of ALS, Sp1-p11-TASK1 dysregulation contributes to increased excitability and vulnerability of motor neurons. Interference with either Sp1 or p11 is neuroprotective, delaying neuron loss and prolonging lifespan in this model. Nitrosative stress, a potential factor in human neurodegeneration, stimulated Sp1 expression and human p11 promoter activity, at least in part, through a Sp1-binding site. Disruption of Sp1 or p11 also has neuroprotective effects in a traumatic model of motor neuron degeneration. Together our work suggests the Sp1-p11-TASK1 pathway is a potential target for treatment of degeneration of motor neurons.


Assuntos
Esclerose Amiotrófica Lateral/patologia , Anexina A2/metabolismo , Neurônios Motores/patologia , Degeneração Neural/patologia , Proteínas do Tecido Nervoso/genética , Canais de Potássio de Domínios Poros em Tandem/genética , Proteínas S100/metabolismo , Fator de Transcrição Sp1/metabolismo , Esclerose Amiotrófica Lateral/etiologia , Animais , Membrana Celular/patologia , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Masculino , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Neurônios Motores/citologia , Degeneração Neural/etiologia , Proteínas do Tecido Nervoso/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Cultura Primária de Células , Regiões Promotoras Genéticas , Ratos , Fator de Transcrição Sp1/genética , Medula Espinal/citologia , Medula Espinal/patologia
14.
Int J Mol Sci ; 20(16)2019 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-31394733

RESUMO

Motor neuron circuitry is one of the most elaborate circuitries in our body, which ensures voluntary and skilled movement that requires cognitive input. Therefore, both the cortex and the spinal cord are involved. The cortex has special importance for motor neuron diseases, in which initiation and modulation of voluntary movement is affected. Amyotrophic lateral sclerosis (ALS) is defined by the progressive degeneration of both the upper and lower motor neurons, whereas hereditary spastic paraplegia (HSP) and primary lateral sclerosis (PLS) are characterized mainly by the loss of upper motor neurons. In an effort to reveal the cellular and molecular basis of neuronal degeneration, numerous model systems are generated, and mouse models are no exception. However, there are many different levels of complexities that need to be considered when developing mouse models. Here, we focus our attention to the upper motor neurons, which are one of the most challenging neuron populations to study. Since mice and human differ greatly at a species level, but the cells/neurons in mice and human share many common aspects of cell biology, we offer a solution by focusing our attention to the affected neurons to reveal the complexities of diseases at a cellular level and to improve translational efforts.


Assuntos
Neurônios Motores/fisiologia , Condução Nervosa , Plasticidade Neuronal , Animais , Modelos Animais de Doenças , Suscetibilidade a Doenças , Ligação Genética , Predisposição Genética para Doença , Humanos , Camundongos , Camundongos Transgênicos , Doença dos Neurônios Motores/etiologia , Doença dos Neurônios Motores/metabolismo , Doença dos Neurônios Motores/fisiopatologia , Neurônios Motores/citologia , Neurônios Motores/ultraestrutura
15.
Physiol Meas ; 40(9): 095002, 2019 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-31470424

RESUMO

Advances in surface electromyographic (EMG) signal decomposition allow researchers to analyze data for 20-50 motor units per contraction. To simplify interpretation, some investigators rely on group mean analysis of the mean firing rate versus recruitment threshold relationship, but it is unclear if this association is linear. OBJECTIVE: To determine whether this relationship is strongest when analyzed via linear, quadratic, or cubic regression. APPROACH: Twenty-one men (mean ± SD age = 24 ± 4 years) and 16 women (21 ± 2 years) performed isometric contractions of the knee extensors at 50% of maximal force while bipolar surface EMG signals were recorded from the vastus lateralis. A decomposition algorithm was used to calculate the mean firing rate and recruitment threshold of each motor unit at accuracy levels ranging from 90.0%-93.0%. Polynominal regression was used to determine if each relationship was best fit with a linear, quadratic, or cubic model. We examined individual contractions and grouped data. MAIN RESULTS: Overall, 80% of the relationships were best fit with a linear model. Quadratic and cubic relationships were more appropriate for 16% and 2% of the contractions, respectively. Selecting varying accuracy levels within a range of 90.0%-93.0% had little influence on whether a given dataset was best fit with a linear, quadratic, or cubic model. Grouping of data provided different relationships than otherwise found on a contraction-by-contraction basis. SIGNIFICANCE: The mean firing rate versus recruitment threshold relationship is typically best fit with a linear model. These relationships should be examined on an individual contraction basis.


Assuntos
Modelos Neurológicos , Neurônios Motores/citologia , Eletromiografia , Feminino , Humanos , Modelos Lineares , Masculino , Contração Muscular/fisiologia , Processamento de Sinais Assistido por Computador , Adulto Jovem
16.
PLoS One ; 14(7): e0218738, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31260485

RESUMO

C. elegans neuronal system constitutes the ideal framework for studying simple, yet realistic, neuronal activity, since the whole nervous system is fully characterized with respect to the exact number of neurons and the neuronal connections. Most recent efforts are devoted to investigate and clarify the signal processing and functional connectivity, which are at the basis of sensing mechanisms, signal transmission, and motor control. In this framework, a refined modelof whole neuron dynamics constitutes a key ingredient to describe the electrophysiological processes, both at thecellular and at the network scale. In this work, we present Hodgkin-Huxley-based models of ion channels dynamics black, built on data available both from C. elegans and from other organisms, expressing homologous channels. We combine these channel models to simulate the electrical activity oftwo among the most studied neurons in C. elegans, which display prototypical dynamics of neuronal activation, the chemosensory AWCON and the motor neuron RMD. Our model properly describes the regenerative responses of the two cells. We analyze in detail the role of ion currents, both in wild type and in in silico knockout neurons. Moreover, we specifically investigate the behavior of RMD, identifying a heterogeneous dynamical response which includes bistable regimes and sustained oscillations. We are able to assess the critical role of T-type calcium currents, carried by CCA-1 channels, and leakage currents in the regulation of RMD response. Overall, our results provide new insights in the activity of key C. elegans neurons. The developed mathematical framework constitute a basis for single-cell and neuronal networks analyses, opening new scenarios in the in silico modeling of C. elegans neuronal system.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Modelos Neurológicos , Neurônios Motores/fisiologia , Rede Nervosa/fisiologia , Células Receptoras Sensoriais/fisiologia , Transmissão Sináptica/fisiologia , Animais , Caenorhabditis elegans/citologia , Proteínas de Caenorhabditis elegans/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/metabolismo , Simulação por Computador , Expressão Gênica , Transporte de Íons , Neurônios Motores/citologia , Rede Nervosa/citologia , Canais de Potássio/genética , Canais de Potássio/metabolismo , Células Receptoras Sensoriais/citologia , Análise de Célula Única/métodos , Canais de Sódio/genética , Canais de Sódio/metabolismo
17.
Dev Biol ; 455(2): 449-457, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31356769

RESUMO

The developing spinal cord builds a boundary between the CNS and the periphery, in the form of a basement membrane. The spinal cord basement membrane is a barrier that retains CNS neuron cell bodies, while being selectively permeable to specific axon types. Spinal motor neuron cell bodies are located in the ventral neural tube next to the floor plate and project their axons out through the basement membrane to peripheral targets. However, little is known about how spinal motor neuron cell bodies are retained inside the ventral neural tube, while their axons can exit. In previous work, we found that disruption of Slit/Robo signals caused motor neuron emigration outside the spinal cord. In the current study, we investigate how Slit/Robo signals are necessary to keep spinal motor neurons within the neural tube. Our findings show that when Slit/Robo signals were removed from motor neurons, they migrated outside the spinal cord. Furthermore, this emigration was associated with abnormal basement membrane protein expression in the ventral spinal cord. Using Robo2 and Slit2 conditional mutants, we found that motor neuron-derived Slit/Robo signals were required to set up a normal basement membrane in the spinal cord. Together, our results suggest that motor neurons produce Slit signals that are required for the basement membrane assembly to retain motor neuron cell bodies within the spinal cord.


Assuntos
Membrana Basal/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/fisiologia , Tubo Neural/citologia , Receptores Imunológicos/fisiologia , Medula Espinal/embriologia , Animais , Movimento Celular , Distroglicanas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/genética , Camundongos , Mutação , Proteínas do Tecido Nervoso/genética , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/fisiologia , Receptores Imunológicos/genética , Transdução de Sinais , Medula Espinal/citologia
18.
Int J Mol Sci ; 20(11)2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31159418

RESUMO

Human tonsil-derived mesenchymal stem cells (T-MSCs) are newly identified MSCs and present typical features of MSCs, including having the differentiation capacity into the three germ layers and excellent proliferation capacity. They are easily sourced and are useful for stem cell therapy in various disease states. We previously reported that T-MSCs could be differentiated into skeletal myocytes and Schwann-like cells; therefore, they are a promising candidate for cell therapies for neuromuscular disease. Motor neurons (MNs), which regulate spontaneous behavior, are affected by a wide range of MN diseases (MNDs) for which there are no effective remedies. We investigated the differentiation potential of MN-like cells derived from T-MSCs (T-MSC-MNCs) for application to therapy of MNDs. After the process of MN differentiation, the expression of MN-related markers, including Islet 1, HB9/HLXB9 (HB9), and choline acetyltransferase (ChAT), was increased when compared with undifferentiated T-MSCs. The secretion of acetylcholine to the conditioned medium was significantly increased after MN differentiation. We cocultured T-MSC-MNCs and human skeletal muscle cells, and confirmed the presence of the acetylcholine receptor clusters, which demonstrated the formation of neuromuscular junctions. The potential functional improvements afforded by these T-MSC-MNCs could be useful in the treatment of MNDs caused by genetic mutation, viral infection, or environmental problems.


Assuntos
Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Neurônios Motores/citologia , Neurônios Motores/fisiologia , Junção Neuromuscular/fisiologia , Tonsila Palatina/citologia , Acetilcolina/metabolismo , Biomarcadores , Células Cultivadas , Expressão Gênica , Humanos , Imuno-Histoquímica , Fibras Musculares Esqueléticas/metabolismo , Fatores de Crescimento Neural/genética , Fatores de Crescimento Neural/metabolismo , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo
19.
Hum Mol Genet ; 28(19): 3175-3187, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31189016

RESUMO

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor nerve cells in the brain and the spinal cord. Etiological mechanisms underlying the disease remain poorly understood; recent studies suggest that deregulation of p25/Cyclin-dependent kinase 5 (Cdk5) activity leads to the hyperphosphorylation of Tau and neurofilament (NF) proteins in ALS transgenic mouse model (SOD1G37R). A Cdk5 involvement in motor neuron degeneration is supported by analysis of three SOD1G37R mouse lines exhibiting perikaryal inclusions of NF proteins and hyperphosphorylation of Tau. Here, we tested the hypothesis that inhibition of Cdk5/p25 hyperactivation in vivo is a neuroprotective factor during ALS pathogenesis by crossing the new transgenic mouse line that overexpresses Cdk5 inhibitory peptide (CIP) in motor neurons with the SOD1G37R, ALS mouse model (TriTg mouse line). The overexpression of CIP in the motor neurons significantly improves motor deficits, extends survival and delays pathology in brain and spinal cord of TriTg mice. In addition, overexpression of CIP in motor neurons significantly delays neuroinflammatory responses in TriTg mouse. Taken together, these data suggest that CIP may serve as a novel therapeutic agent for the treatment of neurodegenerative diseases.


Assuntos
Esclerose Amiotrófica Lateral/terapia , Neurônios Motores/citologia , Proteínas do Tecido Nervoso/genética , Fragmentos de Peptídeos/genética , Esclerose Amiotrófica Lateral/genética , Esclerose Amiotrófica Lateral/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Neurônios Motores/metabolismo , Fenótipo , Fosforilação , Superóxido Dismutase-1/genética , Proteínas tau/metabolismo
20.
Cells ; 8(6)2019 06 13.
Artigo em Inglês | MEDLINE | ID: mdl-31200561

RESUMO

The ubiquitin proteasome system (UPS) plays an important role in regulating numerous cellular processes, and a dysfunctional UPS is thought to contribute to motor neuron disease. Consequently, we sought to map the changing ubiquitome in human iPSCs during their pluripotent stage and following differentiation to motor neurons. Ubiquitinomics analysis identified that spliceosomal and ribosomal proteins were more ubiquitylated in pluripotent stem cells, whilst proteins involved in fatty acid metabolism and the cytoskeleton were specifically ubiquitylated in the motor neurons. The UPS regulator, ubiquitin-like modifier activating enzyme 1 (UBA1), was increased 36-fold in the ubiquitome of motor neurons compared to pluripotent stem cells. Thus, we further investigated the functional consequences of inhibiting the UPS and UBA1 on motor neurons. The proteasome inhibitor MG132, or the UBA1-specific inhibitor PYR41, significantly decreased the viability of motor neurons. Consistent with a role of the UPS in maintaining the cytoskeleton and regulating motor neuron differentiation, UBA1 inhibition also reduced neurite length. Pluripotent stem cells were extremely sensitive to MG132, showing toxicity at nanomolar concentrations. The motor neurons were more resilient to MG132 than pluripotent stem cells but demonstrated higher sensitivity than fibroblasts. Together, this data highlights the important regulatory role of the UPS in pluripotent stem cell survival and motor neuron differentiation.


Assuntos
Diferenciação Celular , Neurônios Motores/citologia , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Ubiquitina/metabolismo , Sobrevivência Celular , Feminino , Fibroblastos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Masculino , Pessoa de Meia-Idade , Proteoma/metabolismo
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