RESUMEN
The protocol describes single-neuron ablation with a 2-photon laser system in the central nervous system (CNS) of intact Drosophila melanogaster larvae. Using this non-invasive method, the developing nervous system can be manipulated in a cell-specific manner. Disrupting the development of individual neurons in a network can be used to study how the nervous system can compensate for the loss of synaptic input. Individual neurons were specifically ablated in the giant fiber system of Drosophila, with a focus on two neurons: the presynaptic giant fiber (GF) and the postsynaptic tergotrochanteral motor neuron (TTMn). The GF synapses with the ipsilateral TTMn, which is crucial to the escape response. Ablating one of the GFs in the 3rd instar brain, just after the GF starts axonal growth, permanently removes the cell during the development of the CNS. The remaining GF reacts to the absent neighbor and forms an ectopic synaptic terminal to the contralateral TTMn. This atypical, bilaterally symmetric terminal innervates both TTMns, as demonstrated by dye coupling, and drives both motor neurons, as demonstrated by electrophysiological assays. In summary, the ablation of a single interneuron demonstrates synaptic competition between a bilateral pair of neurons that can compensate for the loss of one neuron and restore normal responses to the escape circuit.
Asunto(s)
Drosophila melanogaster , Larva , Neuronas Motoras , Sinapsis , Animales , Sinapsis/fisiología , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Terapia por Láser/métodos , Sistema Nervioso Central/citología , Sistema Nervioso Central/fisiología , Neuronas/citología , Neuronas/fisiología , Técnicas de Ablación/métodosRESUMEN
Axonal damage is a common feature of traumatic injury and neurodegenerative disease. The capacity for axons to regenerate and to recover functionality after injury is a phenomenon that is seen readily in the peripheral nervous system, especially in rodent models, but human axonal regeneration is limited and does not lead to full functional recovery. Here we describe a system where dynamics of human axonal outgrowth and regeneration can be evaluated via live imaging of human-induced pluripotent stem cell (hiPSC)-derived neurons cultured in microfluidic systems, in which cell bodies are isolated from their axons. This system could aid in studying axonal outgrowth dynamics and could be useful for testing potential drugs that encourage regeneration and repair of the nervous system.
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Axones , Células Madre Pluripotentes Inducidas , Neuronas Motoras , Regeneración Nerviosa , Humanos , Células Madre Pluripotentes Inducidas/citología , Axones/fisiología , Neuronas Motoras/fisiología , Neuronas Motoras/citología , Regeneración Nerviosa/fisiología , Microfluídica/métodos , Microfluídica/instrumentación , Diferenciación Celular , Células Cultivadas , Dispositivos Laboratorio en un Chip , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/métodos , Técnicas de Cultivo de Célula/métodosRESUMEN
Paralog factors are considered to ensure the robustness of biological processes by providing redundant activity in cells where they are co-expressed. However, the specific contribution of each factor is frequently underestimated. In the developing spinal cord, multiple families of transcription factors successively contribute to differentiate an initially homogenous population of neural progenitors into a myriad of neuronal subsets with distinct molecular, morphological, and functional characteristics. The LIM-homeodomain transcription factors Lhx3, Lhx4, Isl1 and Isl2 promote the segregation and differentiation of spinal motor neurons and V2 interneurons. Based on their high sequence identity and their similar distribution, the Lhx3 and Lhx4 paralogs are considered to contribute similarly to these processes. However, the specific contribution of Lhx4 has never been studied. Here, we provide evidence that Lhx3 and Lhx4 are present in the same cell populations during spinal cord development. Similarly to Lhx3, Lhx4 can form multiproteic complexes with Isl1 or Isl2 and the nuclear LIM interactor NLI. Lhx4 can stimulate a V2-specific enhancer more efficiently than Lhx3 and surpasses Lhx3 in promoting the differentiation of V2a interneurons in chicken embryo electroporation experiments. Finally, Lhx4 inactivation in mice results in alterations of differentiation of the V2a subpopulation, but not of motor neuron production, suggesting that Lhx4 plays unique roles in V2a differentiation that are not compensated by the presence of Lhx3. Thus, Lhx4 could be the major LIM-HD factor involved in V2a interneuron differentiation during spinal cord development and should be considered for in vitro differentiation of spinal neuronal populations.
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Diferenciación Celular , Interneuronas , Proteínas con Homeodominio LIM , Médula Espinal , Factores de Transcripción , Animales , Proteínas con Homeodominio LIM/metabolismo , Proteínas con Homeodominio LIM/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Interneuronas/metabolismo , Interneuronas/citología , Médula Espinal/citología , Médula Espinal/metabolismo , Médula Espinal/embriología , Embrión de Pollo , Ratones , Neuronas Motoras/metabolismo , Neuronas Motoras/citología , Humanos , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Tunicates are the sister group to the vertebrates, yet most species have a life cycle split between swimming larva and sedentary adult phases. During metamorphosis, larval neurons are replaced by adult-specific ones. The regulatory mechanisms underlying this replacement remain largely unknown. Using tissue-specific CRISPR/Cas9-mediated mutagenesis in the tunicate Ciona, we show that orthologs of conserved hindbrain and branchiomeric neuron regulatory factors Pax2/5/8 and Phox2 are required to specify the 'neck', a cellular compartment set aside in the larva to give rise to cranial motor neuron-like neurons post-metamorphosis. Using bulk and single-cell RNA-sequencing analyses, we characterize the transcriptome of the neck downstream of Pax2/5/8. We present evidence that neck-derived adult ciliomotor neurons begin to differentiate in the larva and persist through metamorphosis, contrary to the assumption that the adult nervous system is formed after settlement and the death of larval neurons during metamorphosis. Finally, we show that FGF signaling during the larval phase alters the patterning of the neck and its derivatives. Suppression of FGF converts neck cells into larval neurons that fail to survive metamorphosis, whereas prolonged FGF signaling promotes an adult neural stem cell-like fate.
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Larva , Metamorfosis Biológica , Animales , Larva/crecimiento & desarrollo , Neuronas/metabolismo , Neuronas/citología , Factores de Crecimiento de Fibroblastos/metabolismo , Factores de Crecimiento de Fibroblastos/genética , Regulación del Desarrollo de la Expresión Génica , Neuronas Motoras/metabolismo , Neuronas Motoras/citología , Transducción de Señal/genética , Ciona intestinalis/genética , Supervivencia Celular , Transcriptoma/genética , Factores de Transcripción/metabolismo , Factores de Transcripción/genética , Sistemas CRISPR-Cas/genéticaRESUMEN
A deep understanding of how the brain controls behaviour requires mapping neural circuits down to the muscles that they control. Here, we apply automated tools to segment neurons and identify synapses in an electron microscopy dataset of an adult female Drosophila melanogaster ventral nerve cord (VNC)1, which functions like the vertebrate spinal cord to sense and control the body. We find that the fly VNC contains roughly 45 million synapses and 14,600 neuronal cell bodies. To interpret the output of the connectome, we mapped the muscle targets of leg and wing motor neurons using genetic driver lines2 and X-ray holographic nanotomography3. With this motor neuron atlas, we identified neural circuits that coordinate leg and wing movements during take-off. We provide the reconstruction of VNC circuits, the motor neuron atlas and tools for programmatic and interactive access as resources to support experimental and theoretical studies of how the nervous system controls behaviour.
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Conectoma , Drosophila melanogaster , Neuronas Motoras , Tejido Nervioso , Vías Nerviosas , Sinapsis , Animales , Femenino , Conjuntos de Datos como Asunto , Drosophila melanogaster/anatomía & histología , Drosophila melanogaster/citología , Drosophila melanogaster/fisiología , Drosophila melanogaster/ultraestructura , Extremidades/fisiología , Extremidades/inervación , Holografía , Microscopía Electrónica , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Neuronas Motoras/ultraestructura , Movimiento , Músculos/inervación , Músculos/fisiología , Tejido Nervioso/anatomía & histología , Tejido Nervioso/citología , Tejido Nervioso/fisiología , Tejido Nervioso/ultraestructura , Vías Nerviosas/citología , Vías Nerviosas/fisiología , Vías Nerviosas/ultraestructura , Sinapsis/fisiología , Sinapsis/ultraestructura , Tomografía por Rayos X , Alas de Animales/inervación , Alas de Animales/fisiologíaRESUMEN
The Drosophila larval ventral nerve cord (VNC) shares many similarities with the spinal cord of vertebrates and has emerged as a major model for understanding the development and function of motor systems. Here, we use high-quality scRNA-seq, validated by anatomical identification, to create a comprehensive census of larval VNC cell types. We show that the neural lineages that comprise the adult VNC are already defined, but quiescent, at the larval stage. Using fluorescence-activated cell sorting (FACS)-enriched populations, we separate all motor neuron bundles and link individual neuron clusters to morphologically characterized known subtypes. We discovered a glutamate receptor subunit required for basal neurotransmission and homeostasis at the larval neuromuscular junction. We describe larval glia and endorse the general view that glia perform consistent activities throughout development. This census represents an extensive resource and a powerful platform for future discoveries of cellular and molecular mechanisms in repair, regeneration, plasticity, homeostasis, and behavioral coordination.
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Drosophila melanogaster , Larva , Neuronas Motoras , Animales , Larva/genética , Larva/metabolismo , Neuronas Motoras/metabolismo , Neuronas Motoras/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Neuroglía/metabolismo , Neuroglía/citología , Unión Neuromuscular/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , RNA-Seq/métodos , Análisis de Expresión Génica de una Sola CélulaRESUMEN
BACKGROUND: Spinal cord injury (SCI) is an intractable neurological disease in which functions cannot be permanently restored due to nerve damage. Stem cell therapy is a promising strategy for neuroregeneration after SCI. However, experimental evidence of its therapeutic effect in SCI is lacking. This study aimed to investigate the efficacy of transplanted cells using stepwise combined cell therapy with human mesenchymal stem cells (hMSC) and induced pluripotent stem cell (iPSC)-derived motor neuron progenitor cells (iMNP) in a rat model of SCI. METHODS: A contusive SCI model was developed in Sprague-Dawley rats using multicenter animal spinal cord injury study (MASCIS) impactor. Three protocols were designed and conducted as follows: (Subtopic 1) chronic SCI + iMNP, (Subtopic 2) acute SCI + multiple hMSC injections, and (Main topic) chronic SCI + stepwise combined cell therapy using multiple preemptive hMSC and iMNP. Neurite outgrowth was induced by coculturing hMSC and iPSC-derived motor neuron (iMN) on both two-dimensional (2D) and three-dimensional (3D) spheroid platforms during mature iMN differentiation in vitro. RESULTS: Stepwise combined cell therapy promoted mature motor neuron differentiation and axonal regeneration at the lesional site. In addition, stepwise combined cell therapy improved behavioral recovery and was more effective than single cell therapy alone. In vitro results showed that hMSC and iMN act synergistically and play a critical role in the induction of neurite outgrowth during iMN differentiation and maturation. CONCLUSIONS: Our findings show that stepwise combined cell therapy can induce alterations in the microenvironment for effective cell therapy in SCI. The in vitro results suggest that co-culturing hMSC and iMN can synergistically promote induction of MN neurite outgrowth.
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Diferenciación Celular , Células Madre Pluripotentes Inducidas , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Neuronas Motoras , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal , Traumatismos de la Médula Espinal/terapia , Animales , Células Madre Pluripotentes Inducidas/citología , Trasplante de Células Madre Mesenquimatosas/métodos , Neuronas Motoras/citología , Ratas , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Humanos , Modelos Animales de Enfermedad , Regeneración NerviosaRESUMEN
The unique properties of superparamagnetic iron oxide nanoparticles (SPIONs) enable their use as magnetic biosensors, targeted drug delivery, magnetothermia, magnetic resonance imaging, etc. Today, SPIONs are the only type of metal oxide nanoparticles approved for biomedical application. In this work, we analyzed the cellular response to the previously reported luminescent silica coated SPIONs of the two cell types: M-HeLa cells and primary motor neuron culture. Both internalization pathways and intracellular fate of SPIONs have been compared for these cell lines using fluorescence and transmission electron microscopy. We also applied a pharmacological approach to analyze the endocytosis pathways of SPIONs into the investigated cell lines. The penetration of SPIONs into M-HeLa cells is already noticeable within 30 s of incubation through both caveolin-dependent endocytosis and micropinocytosis. However, incubation for a longer time (1 h at least) is required for the internalization of SPIONs into motor neuron culture cells provided by dynamin-dependent endocytosis and macropinocytosis. The intracellular colocalization assay reveals that the lysosomal internalization pathway of SPIONs is also dependent on the cell type. The lysosomal pathway is much more pronounced for M-HeLa cells compared with motor neurons. The emphasized differences in cellular responses of the two cell lines open up new opportunities in the application of SPIONs in the diagnostics and therapy of cancer cells.
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Endocitosis , Lisosomas , Neuronas Motoras , Dióxido de Silicio , Dióxido de Silicio/química , Dióxido de Silicio/metabolismo , Lisosomas/metabolismo , Humanos , Neuronas Motoras/metabolismo , Neuronas Motoras/citología , Células HeLa , Células Cultivadas , Nanopartículas de Magnetita/química , Animales , Nanopartículas Magnéticas de Óxido de Hierro/químicaRESUMEN
Protein prenylation is one example of a broad class of post-translational modifications where proteins are covalently linked to various hydrophobic moieties. To globally identify and monitor levels of all prenylated proteins in a cell simultaneously, our laboratory and others have developed chemical proteomic approaches that rely on the metabolic incorporation of isoprenoid analogues bearing bio-orthogonal functionality followed by enrichment and subsequent quantitative proteomic analysis. Here, several improvements in the synthesis of the alkyne-containing isoprenoid analogue C15AlkOPP are reported to improve synthetic efficiency. Next, metabolic labeling with C15AlkOPP was optimized to obtain useful levels of metabolic incorporation of the probe in several types of primary cells. Those conditions were then used to study the prenylomes of motor neurons (ES-MNs), astrocytes (ES-As), and their embryonic stem cell progenitors (ESCs), which allowed for the identification of 54 prenylated proteins from ESCs, 50 from ES-MNs, and 84 from ES-As, representing all types of prenylation. Bioinformatic analysis revealed specific enriched pathways, including nervous system development, chemokine signaling, Rho GTPase signaling, and adhesion. Hierarchical clustering showed that most enriched pathways in all three cell types are related to GTPase activity and vesicular transport. In contrast, STRING analysis showed significant interactions in two populations that appear to be cell type dependent. The data provided herein demonstrates that robust incorporation of C15AlkOPP can be obtained in ES-MNs and related primary cells purified via magnetic-activated cell sorting allowing the identification and quantification of numerous prenylated proteins. These results suggest that metabolic labeling with C15AlkOPP should be an effective approach for investigating the role of prenylated proteins in primary cells in both normal cells and disease pathologies, including ALS.
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Alquinos , Astrocitos , Neuronas Motoras , Prenilación de Proteína , Astrocitos/metabolismo , Astrocitos/citología , Animales , Alquinos/química , Alquinos/síntesis química , Neuronas Motoras/metabolismo , Neuronas Motoras/citología , Terpenos/química , Terpenos/síntesis química , Terpenos/metabolismo , Ratones , Estructura Molecular , Células CultivadasRESUMEN
Amyotrophic Lateral Sclerosis (ALS) causes motor neuron degeneration, with 97% of cases exhibiting TDP-43 proteinopathy. Elucidating pathomechanisms has been hampered by disease heterogeneity and difficulties accessing motor neurons. Human induced pluripotent stem cell-derived motor neurons (iPSMNs) offer a solution; however, studies have typically been limited to underpowered cohorts. Here, we present a comprehensive compendium of 429 iPSMNs from 15 datasets, and 271 post-mortem spinal cord samples. Using reproducible bioinformatic workflows, we identify robust upregulation of p53 signalling in ALS in both iPSMNs and post-mortem spinal cord. p53 activation is greatest with C9orf72 repeat expansions but is weakest with SOD1 and FUS mutations. TDP-43 depletion potentiates p53 activation in both post-mortem neuronal nuclei and cell culture, thereby functionally linking p53 activation with TDP-43 depletion. ALS iPSMNs and post-mortem tissue display enrichment of splicing alterations, somatic mutations, and gene fusions, possibly contributing to the DNA damage response.
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Esclerosis Amiotrófica Lateral , Proteínas de Unión al ADN , Inestabilidad Genómica , Transcriptoma , Empalme Alternativo/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Esclerosis Amiotrófica Lateral/patología , Cadáver , Estudios de Cohortes , Conjuntos de Datos como Asunto , Daño del ADN , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Fusión Génica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/citología , Neuronas Motoras/metabolismo , Mutación , Médula Espinal/metabolismo , Transcriptoma/genética , HumanosRESUMEN
The classification of neurons into distinct types reveals hierarchical taxonomic relationships that reflect the extent of similarity between neuronal cell types. At the base of such taxonomies are neuronal cells that are very similar to one another but differ in a small number of reproducible and select features. How are very similar members of a neuron class that share many features instructed to diversify into distinct subclasses? We show here that the six very similar members of the Caenorhabditis elegans IL2 sensory neuron class, which are all specified by a homeobox terminal selector, unc-86/BRN3, differentiate into two subtly distinct subclasses, a dorsoventral subclass and a lateral subclass, by the toggle switch-like action of the sine oculis/SIX homeobox gene unc-39. unc-39 is expressed only in the lateral IL2 neurons, and loss of unc-39 leads to a homeotic transformation of the lateral into the dorsoventral class; conversely, ectopic unc-39 expression converts the dorsoventral subclass into the lateral subclass. Hence, a terminal selector homeobox gene controls both class- as well as subclass-specific features, while a subordinate homeobox gene determines the ability of the class-specific homeobox gene to activate subtype-specific target genes. We find a similar regulatory mechanism operating in a distinct class of six motor neurons. Our findings underscore the importance of homeobox genes in neuronal identity control and invite speculations about homeotic identity transformations as potential drivers of evolutionary novelty during cell-type evolution in the brain.
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Proteínas de Caenorhabditis elegans , Caenorhabditis elegans , Genes Homeobox , Proteínas de Homeodominio , Células Receptoras Sensoriales , Factores de Transcripción , Animales , Caenorhabditis elegans/citología , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/fisiología , Neuronas Motoras/clasificación , Neuronas Motoras/citología , Células Receptoras Sensoriales/clasificación , Células Receptoras Sensoriales/citología , Factores de Transcripción/genética , Factores de Transcripción/fisiologíaRESUMEN
The nervous and immune systems are intricately linked1. Although psychological stress is known to modulate immune function, mechanistic pathways linking stress networks in the brain to peripheral leukocytes remain poorly understood2. Here we show that distinct brain regions shape leukocyte distribution and function throughout the body during acute stress in mice. Using optogenetics and chemogenetics, we demonstrate that motor circuits induce rapid neutrophil mobilization from the bone marrow to peripheral tissues through skeletal-muscle-derived neutrophil-attracting chemokines. Conversely, the paraventricular hypothalamus controls monocyte and lymphocyte egress from secondary lymphoid organs and blood to the bone marrow through direct, cell-intrinsic glucocorticoid signalling. These stress-induced, counter-directional, population-wide leukocyte shifts are associated with altered disease susceptibility. On the one hand, acute stress changes innate immunity by reprogramming neutrophils and directing their recruitment to sites of injury. On the other hand, corticotropin-releasing hormone neuron-mediated leukocyte shifts protect against the acquisition of autoimmunity, but impair immunity to SARS-CoV-2 and influenza infection. Collectively, these data show that distinct brain regions differentially and rapidly tailor the leukocyte landscape during psychological stress, therefore calibrating the ability of the immune system to respond to physical threats.
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Encéfalo , Miedo , Leucocitos , Neuronas Motoras , Vías Nerviosas , Estrés Psicológico , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/inmunología , Encéfalo/citología , Encéfalo/fisiología , COVID-19/inmunología , Quimiocinas/inmunología , Susceptibilidad a Enfermedades , Miedo/fisiología , Glucocorticoides/metabolismo , Humanos , Leucocitos/citología , Leucocitos/inmunología , Linfocitos/citología , Linfocitos/inmunología , Tejido Linfoide/citología , Tejido Linfoide/inmunología , Ratones , Monocitos/citología , Monocitos/inmunología , Neuronas Motoras/citología , Neuronas Motoras/fisiología , Neutrófilos/citología , Neutrófilos/inmunología , Optogenética , Infecciones por Orthomyxoviridae/inmunología , Núcleo Hipotalámico Paraventricular/fisiología , SARS-CoV-2/inmunología , Estrés Psicológico/inmunología , Estrés Psicológico/fisiopatologíaRESUMEN
Background: Biomaterials used as cell growth stimulants should be able to provide adequate cell adhesion with no alteration in cell function. In this work, we developed a three-dimensional model of mouse spinal cord motoneurons on scaffolds composed of electrospun poly-lactic acid (PLA) fibers and plasma-polymerized polypyrrole (PPy)-coated PLA fibers. Methods: The functionality of the cultured motoneurons was assessed by evaluating both the electrophysiological response (i.e., the whole-cell Na+ and K+ currents and the firing of action potentials) and also the expression of the esicular acetylcholine transporter (VAChaT) by immunostaining techniques. While the expression of the VAChaT was confirmed on motoneurons cultured on the fibrous scaffolds, the electrophysiological responses indicated Na+ and K+ currents with lower amplitude and slower action potentials when compared to the response recorded from spinal cord motoneurons cultured on Poly-DL-Ornithine/Laminin- and plasma-polymerized PPy-coated coverslips. Results: From a morphological viewpoint, motoneurons cultured on PLA and PPy-coated PLA scaffolds did not show the development of dendritic and/or axonal processes, which were satisfactorily observed in the bidimensional cultures. Conclusion: We hypothesize that the apparently limited development of dendritic and/or axonal processes could produce a deleterious effect on the electrophysiological response of the cells, which might be due to the limited growth surface available in the fibrous scaffolds and/or to an undesired effect of the purification process.
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Neuronas Motoras , Polímeros , Pirroles , Animales , Células Cultivadas , Fenómenos Electrofisiológicos , Ácido Láctico , Ratones , Neuronas Motoras/citología , Poliésteres/farmacología , Polímeros/farmacología , Andamios del TejidoRESUMEN
We used α-Latrotoxin (α-LTx), the main neurotoxic component of the black widow spider venom, which causes degeneration of the neuromuscular junction (NMJ) followed by a rapid and complete regeneration, as a molecular tool to identify by RNA transcriptomics factors contributing to the structural and functional recovery of the NMJ. We found that Urocortin 2 (UCN2), a neuropeptide involved in the stress response, is rapidly expressed at the NMJ after acute damage and that inhibition of CRHR2, the specific receptor of UCN2, delays neuromuscular transmission rescue. Experiments in neuronal cultures show that CRHR2 localises at the axonal tips of growing spinal motor neurons and that its expression inversely correlates with synaptic maturation. Moreover, exogenous UCN2 enhances the growth of axonal sprouts in cultured neurons in a CRHR2-dependent manner, pointing to a role of the UCN2-CRHR2 axis in the regulation of axonal growth and synaptogenesis. Consistently, exogenous administration of UCN2 strongly accelerates the regrowth of motor axon terminals degenerated by α-LTx, thereby contributing to the functional recovery of neuromuscular transmission after damage. Taken together, our results posit a novel role for UCN2 and CRHR2 as a signalling axis involved in NMJ regeneration.
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Axones/fisiología , Neuronas Motoras/citología , Regeneración Nerviosa , Enfermedades de la Unión Neuromuscular/prevención & control , Unión Neuromuscular/patología , Venenos de Araña/toxicidad , Urocortinas/metabolismo , Animales , Femenino , Ratones , Ratones Endogámicos C57BL , Unión Neuromuscular/efectos de los fármacos , Enfermedades de la Unión Neuromuscular/inducido químicamente , Enfermedades de la Unión Neuromuscular/metabolismo , Enfermedades de la Unión Neuromuscular/patología , Terminales Presinápticos , Ratas , Ratas Sprague-Dawley , Urocortinas/genéticaRESUMEN
Locomotion is a complex behavior required for animal survival. Vertebrate locomotion depends on spinal interneurons termed the central pattern generator (CPG), which generates activity responsible for the alternation of flexor and extensor muscles and the left and right side of the body. It is unknown whether multiple or a single neuronal type is responsible for the control of mammalian locomotion. Here, we show that ventral spinocerebellar tract neurons (VSCTs) drive generation and maintenance of locomotor behavior in neonatal and adult mice. Using mouse genetics, physiological, anatomical, and behavioral assays, we demonstrate that VSCTs exhibit rhythmogenic properties and neuronal circuit connectivity consistent with their essential role in the locomotor CPG. Importantly, optogenetic activation and chemogenetic silencing reveals that VSCTs are necessary and sufficient for locomotion. These findings identify VSCTs as critical components for mammalian locomotion and provide a paradigm shift in our understanding of neural control of complex behaviors.
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Locomoción/fisiología , Mamíferos/fisiología , Neuronas Motoras/citología , Tractos Espinocerebelares/citología , Animales , Axones/fisiología , Fenómenos Electrofisiológicos , Uniones Comunicantes/metabolismo , Silenciador del Gen , Ácido Glutámico/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de Homeodominio/metabolismo , Interneuronas/fisiología , Vértebras Lumbares/metabolismo , Ratones , Propiocepción , Natación , Sinapsis/fisiología , Factores de Transcripción/metabolismoRESUMEN
Transverse sections of the monkey cervical spinal cord from a previous study (Jenny and Inukai, 1983 [1]) were reanalyzed using Neurolucida to create a three-dimensional display of flexor pollicis brevis and abductor pollicis brevis (FAbPBr) motoneurons and dendrites that had been jointly labeled with horse radish peroxidase (HRP). These data were correlated with similar data from a reanalysis of an extensor digitorum communis (EDC) motoneuron pool (Jenny, Cheney, and Jenny, 2018 [2]). The FAbPBr motoneuron columns were located in the C8 (caudal) and T1 segments of the spinal cord and within the most dorsal and medial regions of the motor column pools that innervate hand muscles. Small motoneurons (cell body areas less than 500 µm2 and presumed to be gamma motoneurons) comprised about four percent of the motoneurons and were located throughout the length of the motoneuron pool. HRP labeled dendrites extended radially (360°) from the motoneuron soma but greater numbers of dendrites were directed either dorsomedial to the base of the dorsal horn or medial to the ventromedial gray matter. The longer HRP labeled dendrites and their branch dendrites usually continued in the same radial direction as when originating from the cell body or proximal dendrite. As such we considered the radial direction of the longer HRP labeled dendrites to be a reasonable estimate of the radial direction of the more distal dendritic trees [2]. Both the EDC and FAbPBr motoneuron groups had a greater number of dendrites oriented in dorsal and medial directions from the motoneuron column. Our data continue to suggest that motoneuron dendritic trees have direction-oriented dendrites that extend toward functional terminal regions.
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Vértebras Cervicales/citología , Dendritas/fisiología , Mano/inervación , Neuronas Motoras/fisiología , Animales , Vértebras Cervicales/fisiología , Haplorrinos , Interneuronas/citología , Interneuronas/fisiología , Neuronas Motoras/citología , Músculo Esquelético/inervación , Técnicas de Trazados de Vías Neuroanatómicas , Sinapsis/fisiologíaRESUMEN
Neurons critically rely on the functions of RNA-binding proteins to maintain their polarity and resistance to neurotoxic stress. HnRNP R has a diverse range of post-transcriptional regulatory functions and is important for neuronal development by regulating axon growth. Hnrnpr pre-mRNA undergoes alternative splicing giving rise to a full-length protein and a shorter isoform lacking its N-terminal acidic domain. To investigate functions selectively associated with the full-length hnRNP R isoform, we generated a Hnrnpr knockout mouse (Hnrnprtm1a/tm1a) in which expression of full-length hnRNP R was abolished while production of the truncated hnRNP R isoform was retained. Motoneurons cultured from Hnrnprtm1a/tm1a mice did not show any axonal growth defects but exhibited enhanced accumulation of double-strand breaks and an impaired DNA damage response upon exposure to genotoxic agents. Proteomic analysis of the hnRNP R interactome revealed the multifunctional protein Yb1 as a top interactor. Yb1-depleted motoneurons were defective in DNA damage repair. We show that Yb1 is recruited to chromatin upon DNA damage where it interacts with γ-H2AX, a mechanism that is dependent on full-length hnRNP R. Our findings thus suggest a novel role of hnRNP R in maintaining genomic integrity and highlight the function of its N-terminal acidic domain in this context.
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Cromatina/genética , Daño del ADN , Reparación del ADN/genética , Ribonucleoproteínas Nucleares Heterogéneas/genética , Neuronas Motoras/metabolismo , Proteína 1 de Unión a la Caja Y/genética , Animales , Axones/metabolismo , Línea Celular , Células Cultivadas , Cromatina/metabolismo , Células HEK293 , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Humanos , Immunoblotting , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Motoras/citología , Unión Proteica , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteína 1 de Unión a la Caja Y/metabolismoRESUMEN
The vertebrate neuromuscular junction (NMJ) is formed by a presynaptic motor nerve terminal and a postsynaptic muscle specialization. Cumulative evidence reveals that Wnt ligands secreted by the nerve terminal control crucial steps of NMJ synaptogenesis. For instance, the Wnt3 ligand is expressed by motor neurons at the time of NMJ formation and induces postsynaptic differentiation in recently formed muscle fibers. However, the behavior of presynaptic-derived Wnt ligands at the vertebrate NMJ has not been deeply analyzed. Here, we conducted overexpression experiments to study the expression, distribution, secretion, and function of Wnt3 by transfection of the motor neuron-like NSC-34 cell line and by in ovo electroporation of chick motor neurons. Our findings reveal that Wnt3 is transported along motor axons in vivo following a vesicular-like pattern and reaches the NMJ area. In vitro, we found that endogenous Wnt3 expression increases as the differentiation of NSC-34 cells proceeds. Although NSC-34 cells overexpressing Wnt3 do not modify their morphological differentiation towards a neuronal phenotype, they effectively induce acetylcholine receptor clustering on co-cultured myotubes. These findings support the notion that presynaptic Wnt3 is transported and secreted by motor neurons to induce postsynaptic differentiation in nascent NMJs.
Asunto(s)
Neuronas Motoras/citología , Proteína Wnt3/genética , Proteína Wnt3/metabolismo , Animales , Diferenciación Celular , Línea Celular , Embrión de Pollo , Técnicas de Cocultivo , Electroporación , Ligandos , Ratones , Neuronas Motoras/metabolismo , Unión Neuromuscular/metabolismo , Receptores Colinérgicos/metabolismoRESUMEN
Poliomyelitis-like illness is a common clinical manifestation of neurotropic viral infections. Functional loss and death of motor neurons often lead to reduced muscle tone and paralysis, causing persistent motor sequelae among disease survivors. Despite several reports demonstrating the molecular basis of encephalopathy, the pathogenesis behind virus-induced flaccid paralysis remained largely unknown. The present study for the first time aims to elucidate the mechanism responsible for limb paralysis by studying clinical isolates of Japanese encephalitis virus (JEV) and Chandipura virus (CHPV) responsible for causing acute flaccid paralysis (AFP) in vast regions of Southeast Asia and the Indian subcontinent. An experimental model for studying virus-induced AFP was generated by intraperitoneal injection of 10-day-old BALB/c mice. Progressive decline in motor performance of infected animals was observed, with paralysis being correlated with death of motor neurons (MNs). Furthermore, we demonstrated that upon infection, MNs undergo an extrinsic apoptotic pathway in a RIG-I-dependent fashion via transcription factors pIRF-3 and pIRF-7. Both gene-silencing experiments using specific RIG-I-short interfering RNA and in vivo morpholino abrogated cellular apoptosis, validating the important role of pattern recognition receptor (PRR) RIG-I in MN death. Hence, from our experimental observations, we hypothesize that host innate response plays a significant role in deterioration of motor functioning upon neurotropic virus infections. IMPORTANCE Neurotropic viral infections are an increasingly common cause of immediate or delayed neuropsychiatric sequelae, cognitive impairment, and movement disorders or, in severe cases, death. Given the highest reported disability-adjusted life years and mortality rate worldwide, a better understanding of molecular mechanisms for underlying clinical manifestations like AFP will help in development of more effective tools for therapeutic solutions.
Asunto(s)
Enfermedades Virales del Sistema Nervioso Central/metabolismo , Enfermedades Virales del Sistema Nervioso Central/fisiopatología , Proteína 58 DEAD Box/metabolismo , Virus de la Encefalitis Japonesa (Especie)/fisiología , Neuronas Motoras/citología , Mielitis/metabolismo , Mielitis/fisiopatología , Enfermedades Neuromusculares/metabolismo , Enfermedades Neuromusculares/fisiopatología , Vesiculovirus/fisiología , Animales , Muerte Celular , Enfermedades Virales del Sistema Nervioso Central/genética , Enfermedades Virales del Sistema Nervioso Central/virología , Proteína 58 DEAD Box/genética , Virus de la Encefalitis Japonesa (Especie)/genética , Femenino , Humanos , Factor 3 Regulador del Interferón/genética , Factor 3 Regulador del Interferón/metabolismo , Factor 7 Regulador del Interferón/genética , Factor 7 Regulador del Interferón/metabolismo , Masculino , Ratones , Actividad Motora , Neuronas Motoras/metabolismo , Neuronas Motoras/virología , Mielitis/genética , Mielitis/virología , Enfermedades Neuromusculares/genética , Enfermedades Neuromusculares/virología , Vesiculovirus/genéticaRESUMEN
Generation of patient-derived neurons provides an unprecedented approach in modeling neurological diseases. Here, we describe the direct conversion of adult fibroblasts into motor neurons via lentiviral delivery of transcription factors. Compared with iPSC-based approach, directly converted neurons from donors retain features associated with age, making them ideal systems for modeling age-related neurological diseases. Low yield is the major challenge of this protocol. High quality lentiviruses and optimized cell culture conditions are critical to improve the final yield. For complete details on the use and execution of this protocol, please refer to Ding et al. (2020), Ding et al. (2021), and Liu et al. (2016).