RESUMEN
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease selectively targeting motor neurons in the brain and spinal cord. The reasons for differential motor neuron susceptibility remain elusive. We developed a stem cell-based motor neuron assay to study cell-autonomous mechanisms causing motor neuron degeneration, with implications for ALS. A small-molecule screen identified cyclopiazonic acid (CPA) as a stressor to which stem cell-derived motor neurons were more sensitive than interneurons. CPA induced endoplasmic reticulum stress and the unfolded protein response. Furthermore, CPA resulted in an accelerated degeneration of motor neurons expressing human superoxide dismutase 1 (hSOD1) carrying the ALS-causing G93A mutation, compared to motor neurons expressing wild-type hSOD1. A secondary screen identified compounds that alleviated CPA-mediated motor neuron degeneration: three kinase inhibitors and tauroursodeoxycholic acid (TUDCA), a bile acid derivative. The neuroprotective effects of these compounds were validated in human stem cell-derived motor neurons carrying a mutated SOD1 allele (hSOD1A4V). Moreover, we found that the administration of TUDCA in an hSOD1G93A mouse model of ALS reduced muscle denervation. Jointly, these results provide insights into the mechanisms contributing to the preferential susceptibility of ALS motor neurons, and they demonstrate the utility of stem cell-derived motor neurons for the discovery of new neuroprotective compounds.
Asunto(s)
Neuronas Motoras/citología , Células Madre/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Estrés del Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/genética , Humanos , Indoles/farmacología , Ratones , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Mutación , Células Madre/efectos de los fármacos , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Ácido Tauroquenodesoxicólico/farmacologíaRESUMEN
Air breathing is an essential motor function for vertebrates living on land. The rhythm that drives breathing is generated within the central nervous system and relayed via specialised subsets of spinal motor neurons to muscles that regulate lung volume. In mammals, a key respiratory muscle is the diaphragm, which is innervated by motor neurons in the phrenic nucleus. Remarkably, relatively little is known about how this crucial subtype of motor neuron is generated during embryogenesis. Here, we used direct differentiation of motor neurons from mouse embryonic stem cells as a tool to identify genes that direct phrenic neuron identity. We find that three determinants, Pou3f1, Hoxa5 and Notch, act in combination to promote a phrenic neuron molecular identity. We show that Notch signalling induces Pou3f1 in developing motor neurons in vitro and in vivo. This suggests that the phrenic neuron lineage is established through a local source of Notch ligand at mid-cervical levels. Furthermore, we find that the cadherins Pcdh10, which is regulated by Pou3f1 and Hoxa5, and Cdh10, which is controlled by Pou3f1, are both mediators of like-like clustering of motor neuron cell bodies. This specific Pcdh10/Cdh10 activity might provide the means by which phrenic neurons are assembled into a distinct nucleus. Our study provides a framework for understanding how phrenic neuron identity is conferred and will help to generate this rare and inaccessible yet vital neuronal subtype directly from pluripotent stem cells, thus facilitating subsequent functional investigations.
Asunto(s)
Cadherinas/metabolismo , Células Madre Embrionarias/citología , Neuronas Motoras/citología , Factor 6 de Transcripción de Unión a Octámeros/metabolismo , Nervio Frénico/embriología , Transducción de Señal/fisiología , Animales , Diferenciación Celular/fisiología , Diafragma/inervación , Citometría de Flujo , Proteínas de Homeodominio/metabolismo , Ratones , Neuronas Motoras/fisiología , Fosfoproteínas/metabolismo , Nervio Frénico/citología , Protocadherinas , Reacción en Cadena en Tiempo Real de la Polimerasa , Receptores Notch/metabolismo , Transducción de Señal/genética , Factores de Transcripción , TranscriptomaRESUMEN
V3 spinal interneurons are a key element of the spinal circuits, which control motor function. However, to date, there are no effective ways of deriving a pure V3 population from human pluripotent stem cells. Here, we report a method for differentiation and isolation of spinal V3 interneurons, combining extrinsic factor-mediated differentiation and magnetic activated cell sorting. We found that differentiation of V3 progenitors can be enhanced with a higher concentration of Sonic Hedgehog agonist, as well as culturing cells in 3D format. To enable V3 progenitor purification from mixed differentiation cultures, we developed a transgene reporter, with a part of the regulatory region of V3-specific gene Nkx2-2 driving the expression of a membrane marker CD14. We found that in human cells, NKX2-2 initially exhibited co-labelling with motor neuron progenitor marker, but V3 specificity emerged as the differentiation culture progressed. At these later differentiation timepoints, we were able to enrich V3 progenitors labelled with CD14 to ~ 95% purity, and mature them to postmitotic V3 interneurons. This purification tool for V3 interneurons will be useful for in vitro disease modeling, studies of normal human neural development and potential cell therapies for disorders of the spinal cord.
Asunto(s)
Células Madre Embrionarias Humanas , Humanos , Diferenciación Celular , Proteínas Hedgehog/metabolismo , Interneuronas/metabolismo , Neuronas Motoras/metabolismo , Médula Espinal/metabolismo , Proteína Homeobox Nkx-2.2/genéticaRESUMEN
Many devastating neuromuscular diseases currently lack effective treatments. This is in part due to a lack of drug discovery platforms capable of assessing complex human neuromuscular disease phenotypes in a scalable manner. A major obstacle has been generating scaffolds to stabilise mature contractile myofibers in a multi-well assay format amenable to high content image (HCI) analysis. This study describes the development of a scalable human induced pluripotent stem cell (iPSC)-neuromuscular disease model, whereby suspended elastomer nanofibers support long-term stability, alignment, maturation, and repeated contractions of iPSC-myofibers, innervated by iPSC-motor neurons in 96-well assay plates. In this platform, optogenetic stimulation of the motor neurons elicits robust myofiber-contractions, providing a functional readout of neuromuscular transmission. Additionally, HCI analysis provides rapid and automated quantification of axonal outgrowth, myofiber morphology, and neuromuscular synapse number and morphology. By incorporating amyotrophic lateral sclerosis (ALS)-related TDP-43G298Smutant motor neurons and CRISPR-corrected controls, key neuromuscular disease phenotypes are recapitulated, including weaker myofiber contractions, reduced axonal outgrowth, and reduced number of neuromuscular synapses. Treatment with a candidate ALS drug, the receptor-interacting protein kinase-1 (RIPK1)-inhibitor necrostatin-1, rescues these phenotypes in a dose-dependent manner, highlighting the potential of this platform to screen novel treatments for neuromuscular diseases.
Asunto(s)
Esclerosis Amiotrófica Lateral , Células Madre Pluripotentes Inducidas , Nanofibras , Enfermedades Neuromusculares , Humanos , ElastómerosRESUMEN
Human neuromuscular diseases represent a diverse group of disorders with unmet clinical need, ranging from muscular dystrophies, such as Duchenne muscular dystrophy (DMD), to neurodegenerative disorders, such as amyotrophic lateral sclerosis (ALS). In many of these conditions, axonal and neuromuscular synapse dysfunction have been implicated as crucial pathological events, highlighting the need for in vitro disease models that accurately recapitulate these aspects of human neuromuscular physiology. The protocol reported here describes the co-culture of neural spheroids composed of human pluripotent stem cell (PSC)-derived motor neurons and astrocytes, and human PSC-derived myofibers in 3D compartmentalised microdevices to generate functional human neuromuscular circuits in vitro. In this microphysiological model, motor axons project from a central nervous system (CNS)-like compartment along microchannels to innervate skeletal myofibers plated in a separate muscle compartment. This mimics the spatial organization of neuromuscular circuits in vivo. Optogenetics, particle image velocimetry (PIV) analysis, and immunocytochemistry are used to control, record, and quantify functional neuromuscular transmission, axonal outgrowth, and neuromuscular synapse number and morphology. This approach has been applied to study disease-specific phenotypes for DMD and ALS by incorporating patient-derived and CRISPR-corrected human PSC-derived motor neurons and skeletal myogenic progenitors into the model, as well as testing candidate drugs for rescuing pathological phenotypes. The main advantages of this approach are: i) its simple design; ii) the in vivo-like anatomical separation between CNS and peripheral muscle; and iii) the amenability of the approach to high power imaging. This opens up the possibility for carrying out live axonal transport and synaptic imaging assays in future studies, in addition to the applications reported in this study. Graphical abstract Graphical abstract abbreviations: Channelrhodopsin-2 (CHR2+), pluripotent stem cell (PSC), motor neurons (MNs), myofibers (MFs), neuromuscular junction (NMJ).
RESUMEN
Dysregulated neuronal excitability is a hallmark of amyotrophic lateral sclerosis (ALS). We sought to investigate how functional changes to the axon initial segment (AIS), the site of action potential generation, could impact neuronal excitability in ALS human induced pluripotent stem cell (hiPSC) motor neurons. We find that early TDP-43 and C9orf72 hiPSC motor neurons show an increase in the length of the AIS and impaired activity-dependent AIS plasticity that is linked to abnormal homeostatic regulation of neuronal activity and intrinsic hyperexcitability. In turn, these hyperactive neurons drive increased spontaneous myofiber contractions of in vitro hiPSC motor units. In contrast, late hiPSC and postmortem ALS motor neurons show AIS shortening, and hiPSC motor neurons progress to hypoexcitability. At a molecular level, aberrant expression of the AIS master scaffolding protein ankyrin-G and AIS-specific voltage-gated sodium channels mirror these dynamic changes in AIS function and excitability. Our results point toward the AIS as an important site of dysfunction in ALS motor neurons.
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Esclerosis Amiotrófica Lateral , Segmento Inicial del Axón , Células Madre Pluripotentes Inducidas , Humanos , Segmento Inicial del Axón/metabolismo , Esclerosis Amiotrófica Lateral/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Neuronas Motoras/metabolismo , Potenciales de Acción/fisiologíaRESUMEN
Bardet-Biedl syndrome (BBS) is a ciliopathy with pleiotropic effects on multiple tissues, including the kidney. Here we have compared renal differentiation of iPS cells from healthy and BBS donors. High content image analysis of WT1-expressing kidney progenitors showed that cell proliferation, differentiation and cell shape were similar in healthy, BBS1, BBS2, and BBS10 mutant lines. We then examined three patient lines with BBS10 mutations in a 3D kidney organoid system. The line with the most deleterious mutation, with low BBS10 expression, expressed kidney marker genes but failed to generate 3D organoids. The other two patient lines expressed near normal levels of BBS10 mRNA and generated multiple kidney lineages within organoids when examined at day 20 of organoid differentiation. However, on prolonged culture (day 27) the proximal tubule compartment degenerated. Introducing wild type BBS10 into the most severely affected patient line restored organoid formation, whereas CRISPR-mediated generation of a truncating BBS10 mutation in a healthy line resulted in failure to generate organoids. Our findings provide a basis for further mechanistic studies of the role of BBS10 in the kidney.
RESUMEN
Anxiety disorders are the most prevalent mental disorders in developed societies. Although roles for the prefrontal cortex, amygdala, hippocampus and mediodorsal thalamus in anxiety disorders are well documented, molecular mechanisms contributing to the functions of these structures are poorly understood. Here we report that deletion of Lynx2, a mammalian prototoxin gene that is expressed at high levels in anxiety associated brain areas, results in elevated anxiety-like behaviors. We show that LYNX2 can bind to and modulate neuronal nicotinic receptors, and that loss of Lynx2 alters the actions of nicotine on glutamatergic signaling in the prefrontal cortex. Our data identify Lynx2 as an important component of the molecular mechanisms that control anxiety, and suggest that altered glutamatergic signaling in the prefrontal cortex of Lynx2 mutant mice contributes to increased anxiety-related behaviors.
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Ansiedad , Conducta Animal , Glicoproteínas de Membrana/fisiología , Neuropéptidos/fisiología , Animales , Trastornos de Ansiedad/etiología , Ácido Glutámico , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Ratones Mutantes , Neuropéptidos/genética , Neuropéptidos/metabolismo , Unión Proteica , Receptores Nicotínicos/metabolismo , Transmisión SinápticaRESUMEN
Generating skeletal muscle tissue that mimics the cellular alignment, maturation, and function of native skeletal muscle is an ongoing challenge in disease modeling and regenerative therapies. Skeletal muscle cultures require extracellular guidance and mechanical support to stabilize contractile myofibers. Existing microfabrication-based solutions are limited by complex fabrication steps, low throughput, and challenges in measuring dynamic contractile function. Here, the synthesis and characterization of a new biobased nanohybrid elastomer, which is electrospun into aligned nanofiber sheets to mimic the skeletal muscle extracellular matrix, is presented. The polymer exhibits remarkable hyperelasticity well-matched to that of native skeletal muscle (≈11-50 kPa), with ultimate strain ≈1000%, and elastic modulus ≈25 kPa. Uniaxially aligned nanofibers guide myoblast alignment, enhance sarcomere formation, and promote a ≈32% increase in myotube fusion and ≈50% increase in myofiber maturation. The elastomer nanofibers stabilize optogenetically controlled human induced pluripotent stem cell derived skeletal myofibers. When activated by blue light, the myofiber-nanofiber hybrid constructs maintain a significantly higher (>200%) contraction velocity and specific force (>280%) compared to conventional culture methods. The engineered myofibers exhibit a power density of ≈35 W m-3 . This system is a promising new skeletal muscle tissue model for applications in muscular disease modeling, drug discovery, and muscle regeneration.
Asunto(s)
Células Madre Pluripotentes Inducidas , Nanofibras , Diferenciación Celular , Elastómeros , Humanos , Fibras Musculares Esqueléticas , Músculo Esquelético , Ingeniería de Tejidos/métodos , Andamios del TejidoRESUMEN
Duchenne muscular dystrophy (DMD) is caused by dystrophin gene mutations leading to skeletal muscle weakness and wasting. Dystrophin is enriched at the neuromuscular junction (NMJ), but how NMJ abnormalities contribute to DMD pathogenesis remains unclear. Here, we combine transcriptome analysis and modeling of DMD patient-derived neuromuscular circuits with CRISPR-corrected isogenic controls in compartmentalized microdevices. We show that NMJ volumes and optogenetic motor neuronstimulated myofiber contraction are compromised in DMD neuromuscular circuits, which can be rescued by pharmacological inhibition of TGFß signaling, an observation validated in a 96-well human neuromuscular circuit coculture assay. These beneficial effects are associated with normalization of dysregulated gene expression in DMD myogenic transcriptomes affecting NMJ assembly (e.g., MUSK) and axon guidance (e.g., SLIT2 and SLIT3). Our study provides a new human microphysiological model for investigating NMJ defects in DMD and assessing candidate drugs and suggests that enhancing neuromuscular connectivity may be an effective therapeutic strategy.
RESUMEN
Dendritic cells (DCs)(*) fulfill an important regulatory function at the interface of the innate and adaptive immune system. The thymus and activation-regulated chemokine (TARC/CCL17) is produced by DCs and facilitates the attraction of activated T cells. Using a fluorescence-based in vivo reporter system, we show that CCL17 expression in mice is found in activated Langerhans cells and mature DCs located in various lymphoid and nonlymphoid organs, and is up-regulated after stimulation with Toll-like receptor ligands. DCs expressing CCL17 belong to the CD11b(+)CD8(-)Dec205(+) DC subset, including the myeloid-related DCs located in the subepithelial dome of Peyer's patches. CCL17-deficient mice mount diminished T cell-dependent contact hypersensitivity responses and display a deficiency in rejection of allogeneic organ transplants. In contrast to lymphoid organs located at external barriers of the skin and mucosa, CCL17 is not expressed in the spleen, even after systemic microbial challenge or after in vitro stimulation. These findings indicate that CCL17 production is a hallmark of local DC stimulation in peripheral organs but is absent from the spleen as a filter of blood-borne antigens.
Asunto(s)
Quimiocinas CC/biosíntesis , Células Dendríticas/metabolismo , Bazo/metabolismo , Animales , Antígeno CD11c/metabolismo , Quimiocina CCL17 , Quimiocinas CC/genética , Quimiocinas CC/inmunología , Células Dendríticas/inmunología , Dermatitis por Contacto/inmunología , Células Epidérmicas , Epidermis/inmunología , Epidermis/metabolismo , Marcación de Gen , Genes Reporteros , Supervivencia de Injerto , Proteínas Fluorescentes Verdes , Trasplante de Corazón , Células de Langerhans/inmunología , Células de Langerhans/metabolismo , Lipopolisacáridos/inmunología , Listeriosis/inmunología , Proteínas Luminiscentes/metabolismo , Tejido Linfoide/citología , Tejido Linfoide/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fagocitosis , Bazo/citología , Bazo/inmunologíaRESUMEN
Motor neurons, alone among neurons in the vertebrate CNS, extend axons out of the neural tube to innervate peripheral targets. Two classes of motor neurons, termed vMNs and dMNs, extend axons out of the neural tube via ventral and dorsal exit points, respectively, in accord with their homeodomain transcription factor repertoire. Downstream of these transcriptional codes, the cell surface receptors that shape initial motor axon trajectories have not been identified. We show here that the chemokine receptor Cxcr4 is expressed on the axons of vMNs as they follow their ventral trajectory, whereas its ligand, Cxcl12, is expressed by mesenchymal cells surrounding the ventral neural tube. Genetic studies reveal that Cxcl12-Cxcr4 signaling directs the ventral trajectory of spinal vMNs. In its absence, these neurons adopt a dMN-like trajectory, despite preservation of their vMN transcriptional identity. Thus, the status of Cxcr4 signaling helps to determine the initial axonal trajectory of mammalian motor neurons.
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Quimiocinas CXC/fisiología , Quimiocinas/fisiología , Neuronas Motoras/fisiología , Transducción de Señal/fisiología , Animales , Axones/fisiología , Movimiento Celular , Células Cultivadas , Quimiocina CXCL12 , Quimiocinas CXC/biosíntesis , Quimiocinas CXC/genética , Citometría de Flujo , Regulación de la Expresión Génica , Conos de Crecimiento/fisiología , Inmunohistoquímica , Hibridación in Situ , Ratones , Mutación/fisiología , ARN Mensajero/biosíntesis , Rombencéfalo/citología , Rombencéfalo/fisiología , Médula Espinal/citologíaRESUMEN
Neuropilins, secreted semaphorin coreceptors, are expressed in discrete populations of spinal motor neurons, suggesting they provide critical guidance information for the establishment of functional motor circuitry. We show here that motor axon growth and guidance are impaired in the absence of Sema3A-Npn-1 signaling. Motor axons enter the limb precociously, showing that Sema3A controls the timing of motor axon in-growth to the limb. Lateral motor column (LMC) motor axons within spinal nerves are defasciculated as they grow toward the limb and converge in the plexus region. Medial and lateral LMC motor axons show dorso-ventral guidance defects in the forelimb. In contrast, Sema3F-Npn-2 signaling guides the axons of a medial subset of LMC neurons to the ventral limb, but plays no major role in regulating their fasciculation. Thus, Sema3A-Npn-1 and Sema3F-Npn-2 signaling control distinct steps of motor axon growth and guidance during the formation of spinal motor connections.
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Conos de Crecimiento/metabolismo , Neuronas Motoras/metabolismo , Neuropilinas/metabolismo , Semaforinas/metabolismo , Transducción de Señal/fisiología , Médula Espinal/embriología , Animales , Tipificación del Cuerpo/fisiología , Plexo Braquial/embriología , Diferenciación Celular/fisiología , Embrión de Pollo , Miembro Anterior/embriología , Miembro Anterior/inervación , Regulación del Desarrollo de la Expresión Génica/fisiología , Conos de Crecimiento/ultraestructura , Miembro Posterior/embriología , Miembro Posterior/inervación , Esbozos de los Miembros/embriología , Esbozos de los Miembros/inervación , Plexo Lumbosacro/embriología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Neuronas Motoras/citología , Músculo Esquelético/embriología , Músculo Esquelético/inervación , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Neuropilina-1/genética , Neuropilina-1/metabolismo , Neuropilina-2/genética , Neuropilina-2/metabolismo , Semaforina-3A/genética , Semaforina-3A/metabolismo , Médula Espinal/citología , Médula Espinal/metabolismoRESUMEN
Motor neurons project axons from the hindbrain and spinal cord to muscle, where they induce myofibre contractions through neurotransmitter release at neuromuscular junctions. Studies of neuromuscular junction formation and homeostasis have been largely confined to in vivo models. In this study we have merged three powerful tools - pluripotent stem cells, optogenetics and microfabrication - and designed an open microdevice in which motor axons grow from a neural compartment containing embryonic stem cell-derived motor neurons and astrocytes through microchannels to form functional neuromuscular junctions with contractile myofibers in a separate compartment. Optogenetic entrainment of motor neurons in this reductionist neuromuscular circuit enhanced neuromuscular junction formation more than two-fold, mirroring the activity-dependence of synapse development in vivo. We incorporated an established motor neuron disease model into our system and found that coculture of motor neurons with SOD1G93A astrocytes resulted in denervation of the central compartment and diminished myofiber contractions, a phenotype which was rescued by the Receptor Interacting Serine/Threonine Kinase 1 (RIPK1) inhibitor Necrostatin. This coculture system replicates key aspects of nerve-muscle connectivity in vivo and represents a rapid and scalable alternative to animal models of neuromuscular function and disease.
RESUMEN
In developing neurons, phosphoinositide 3-kinases (PI3Ks) control axon growth and branching by positively regulating PI3K/PI(3,4,5)P3, but how neurons are able to generate sufficient PI(3,4,5)P3 in the presence of high levels of the antagonizing phosphatase PTEN is difficult to reconcile. We find that normal axon morphogenesis involves homeostasis of elongation and branch growth controlled by accumulation of PI(3,4,5)P3 through PTEN inhibition. We identify a plasma membrane-localized protein-protein interaction of PTEN with plasticity-related gene 2 (PRG2). PRG2 stabilizes membrane PI(3,4,5)P3 by inhibiting PTEN and localizes in nanoclusters along axon membranes when neurons initiate their complex branching behavior. We demonstrate that PRG2 is both sufficient and necessary to account for the ability of neurons to generate axon filopodia and branches in dependence on PI3K/PI(3,4,5)P3 and PTEN. Our data indicate that PRG2 is part of a neuronal growth program that induces collateral branch growth in axons by conferring local inhibition of PTEN.
Asunto(s)
Axones/metabolismo , Proteínas de la Membrana/metabolismo , Fosfohidrolasa PTEN/metabolismo , Animales , Células COS , Chlorocebus aethiops , Femenino , Humanos , Masculino , Proteínas de la Membrana/genética , Ratones , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/genética , Fosfatos de Fosfatidilinositol/metabolismoRESUMEN
The properties of mammalian spinal interneurons that underlie rhythmic locomotor networks remain poorly described. Using postnatal transgenic mice in which expression of green fluorescent protein is driven by the promoter for the homeodomain transcription factor Hb9, as well as Hb9-lacZ knock-in mice, we describe a novel population of glutamatergic interneurons located adjacent to the ventral commissure from cervical to midlumbar spinal cord levels. Hb9+ interneurons exhibit strong postinhibitory rebound and demonstrate pronounced membrane potential oscillations in response to chemical stimuli that induce locomotor activity. These data provide a molecular and physiological delineation of a small population of ventral spinal interneurons that exhibit homogeneous electrophysiological features, the properties of which suggest that they are candidate locomotor rhythm-generating interneurons.
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Proteínas de Homeodominio/genética , Interneuronas/fisiología , Médula Espinal/fisiología , Factores de Transcripción/genética , Animales , Bovinos , Electrofisiología , Regulación de la Expresión Génica , Proteínas Fluorescentes Verdes/genética , Hormona del Crecimiento/genética , Inmunohistoquímica , Hibridación in Situ , Ratones , Ratones Transgénicos , Regiones Promotoras Genéticas , Empalme del ARN , Recombinación Genética , beta-Galactosidasa/genéticaRESUMEN
Controlling muscle function is essential for human behaviour and survival, thus, impairment of motor function and muscle paralysis can severely impact quality of life and may be immediately life-threatening, as occurs in many cases of traumatic spinal cord injury (SCI) and in patients with amyotrophic lateral sclerosis (ALS). Repairing damaged spinal motor circuits, in either SCI or ALS, currently remains an elusive goal. Therefore alternative strategies are needed to artificially control muscle function and thereby enable essential motor tasks. This review focuses on recent advances towards restoring motor function, with a particular focus on stem cell-derived neuronal engraftment strategies, optogenetic control of motor function and the potential future translational application of these approaches.
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Esclerosis Amiotrófica Lateral/terapia , Actividad Motora/fisiología , Células-Madre Neurales/trasplante , Optogenética/métodos , Recuperación de la Función , Traumatismos de la Médula Espinal/terapia , Animales , HumanosRESUMEN
PTEN is a lipid and protein phosphatase that regulates a diverse range of cellular mechanisms. PTEN is mainly present in the cytosol and transiently associates with the plasma membrane to dephosphorylate PI(3,4,5)P3, thereby antagonizing the PI3-Kinase signaling pathway. Recently, PTEN has been shown to associate also with organelles such as the endoplasmic reticulum (ER), the mitochondria, or the nucleus, and to be secreted outside of the cell. In addition, PTEN dynamically localizes to specialized sub-cellular compartments such as the neuronal growth cone or dendritic spines. The diverse localizations of PTEN imply a tight temporal and spatial regulation, orchestrated by mechanisms such as posttranslational modifications, formation of distinct protein-protein interactions, or the activation/recruitment of PTEN downstream of external cues. The regulation of PTEN function is thus not only important at the enzymatic activity level, but is also associated to its spatial distribution. In this review we will summarize (i) recent findings that highlight mechanisms controlling PTEN movement and sub-cellular localization, and (ii) current understanding of how PTEN localization is achieved by mechanisms controlling posttranslational modification, by association with binding partners and by PTEN structural or activity requirements. Finally, we will discuss the possible roles of compartmentalized PTEN in developing and mature neurons in health and disease.
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Damage to the central nervous system caused by traumatic injury or neurological disorders can lead to permanent loss of voluntary motor function and muscle paralysis. Here, we describe an approach that circumvents central motor circuit pathology to restore specific skeletal muscle function. We generated murine embryonic stem cell-derived motor neurons that express the light-sensitive ion channel channelrhodopsin-2, which we then engrafted into partially denervated branches of the sciatic nerve of adult mice. These engrafted motor neurons not only reinnervated lower hind-limb muscles but also enabled their function to be restored in a controllable manner using optogenetic stimulation. This synthesis of regenerative medicine and optogenetics may be a successful strategy to restore muscle function after traumatic injury or disease.
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Luz , Neuronas Motoras/fisiología , Neuronas Motoras/trasplante , Músculo Esquelético/inervación , Músculo Esquelético/fisiología , Optogenética , Animales , Axones/fisiología , Línea Celular , Channelrhodopsins , Estimulación Eléctrica , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Femenino , Miembro Posterior , Contracción Isométrica , Ratones , Ratones Endogámicos C57BL , Neuronas Motoras/citología , Desnervación Muscular , Fibras Musculares Esqueléticas/fisiología , Regeneración Nerviosa , Nervio Ciático/fisiología , Transfección , TransgenesRESUMEN
The development of different brain regions involves the coordinated control of proliferation and cell fate specification along and across the neuraxis. Here, we identify Plxdc2 as a novel regulator of these processes, using in ovo electroporation and in vitro cultures of mammalian cells. Plxdc2 is a type I transmembrane protein with some homology to nidogen and to plexins. It is expressed in a highly discrete and dynamic pattern in the developing nervous system, with prominent expression in various patterning centres. In the chick neural tube, where Plxdc2 expression parallels that seen in the mouse, misexpression of Plxdc2 increases proliferation and alters patterns of neurogenesis, resulting in neural tube thickening at early stages. Expression of the Plxdc2 extracellular domain alone, which can be cleaved and shed in vivo, is sufficient for this activity, demonstrating a cell non-autonomous function. Induction of proliferation is also observed in cultured embryonic neuroepithelial cells (ENCs) derived from E9.5 mouse neural tube, which express a Plxdc2-binding activity. These experiments uncover a direct molecular activity of Plxdc2 in the control of proliferation, of relevance in understanding the role of this protein in various cancers, where its expression has been shown to be altered. They also implicate Plxdc2 as a novel component of the network of signalling molecules known to coordinate proliferation and differentiation in the developing nervous system.