Co-Culture of ES Cell-Derived Motor Neurons and Myotubes Show the Formation of Neuromuscular Junctions and Changes in Gene Expression

Cabeza (caz) is the single Drosophila melanogaster orthologue of the human FET proteins FUS, TAF15, and EWSR1, which have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. In this study, we identified Xrp1, a nuclear chromatin-binding protein, as a key modifier of caz mutant phenotypes. Xrp1 expression was strongly up-regulated in caz mutants, and Xrp1 heterozygosity rescued their motor defects and life span. Interestingly, selective neuronal Xrp1 knockdown was sufficient to rescue, and neuronal Xrp1 overexpression phenocopied caz mutant phenotypes. The caz/Xrp1 genetic interaction depended on the functionality of the AT-hook DNA-binding domain in Xrp1, and the majority of Xrp1-interacting proteins are involved in gene expression regulation. Consistently, caz mutants displayed gene expression dysregulation, which was mitigated by Xrp1 heterozygosity. Finally, Xrp1 knockdown substantially rescued the motor deficits and life span of flies expressing ALS mutant FUS in motor neurons, implicating gene expression dysregulation in ALS-FUS pathogenesis.

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Mouse embryonic stem cells can differentiate via multiple paths to the same state

eLife ◽

10.7554/elife.26945 ◽

2017 ◽

Vol 6 ◽

Cited By ~ 31

Author(s):

James Alexander Briggs ◽

Victor C Li ◽

Seungkyu Lee ◽

Clifford J Woolf ◽

Allon Klein ◽

...

Keyword(s):

Gene Expression ◽

Motor Neuron ◽

Motor Neurons ◽

Embryonic Stem ◽

The Novel ◽

Transitional State ◽

Single Cell Rna Sequencing ◽

Intermediate States ◽

Multiple Paths ◽

Alternative Routes

In embryonic development, cells differentiate through stereotypical sequences of intermediate states to generate particular mature fates. By contrast, driving differentiation by ectopically expressing terminal transcription factors (direct programming) can generate similar fates by alternative routes. How differentiation in direct programming relates to embryonic differentiation is unclear. We applied single-cell RNA sequencing to compare two motor neuron differentiation protocols: a standard protocol approximating the embryonic lineage, and a direct programming method. Both initially undergo similar early neural commitment. Later, the direct programming path diverges into a novel transitional state rather than following the expected embryonic spinal intermediates. The novel state in direct programming has specific and uncharacteristic gene expression. It forms a loop in gene expression space that converges separately onto the same final motor neuron state as the standard path. Despite their different developmental histories, motor neurons from both protocols structurally, functionally, and transcriptionally resemble motor neurons isolated from embryos.

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Myomirnas Role in Als Suggest a Crosstalk between Muscle and Motor Neuron

10.20944/preprints201810.0709.v1 ◽

2018 ◽

Author(s):

Valentina Pegoraro ◽

Antonio Merico ◽

Corrado Angelini

Keyword(s):

Skeletal Muscle ◽

Motor Neuron ◽

Aerobic Exercise ◽

Muscle Atrophy ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Neurodegenerative Disorder ◽

Disease Process ◽

Muscle Fibres ◽

Wide Range

Amyotrophic lateral sclerosis (ALS) is a rare, progressive, neurodegenerative disorder caused by degeneration of upper and lower motor neurons. The disease process leads from lower motor neuron involvement to progressive muscle atrophy, weakness, fasciculations for the upper motor neuron involvement to spasticity. Muscle atrophy in ALS is caused by a dysregulation in the molecular network controlling fast and slow muscle fibres. Denervation and reinnervation processes in skeletal muscle occur in the course of ALS and are modulated by rehabilitation. MicroRNAs (miRNAs) are small non-coding RNAs that modulate a wide range of biological functions under various pathophysiological conditions. MiRNAs can be secreted by various cell types and they are markedly stable in body fluids. MiR-1, miR-133 a, miR-133b, and miR-206 are called “myomiRs” and are considered markers of myogenesis during muscle regeneration and neuromuscular junction stabilization or sprouting. We observed a positive effect of a standard aerobic exercise rehabilitative protocol conducted for six weeks in 18 ALS patients during hospitalization in our center. We correlated clinical scales with molecular data on myomiRs. After six weeks of moderate aerobic exercise, myomiRNAs were down-regulated, suggesting an active proliferation of satellite cells in muscle and increased neuromuscular junctions. Our data suggest that circulating miRNAs modulate during skeletal muscle recovery in response to physical rehabilitation in ALS.

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Single cell transcriptome profiling of the human developing spinal cord reveals a conserved genetic programme with human specific features

10.1101/2021.04.12.439474 ◽

2021 ◽

Author(s):

Teresa Rayon ◽

Rory J. Maizels ◽

Christopher Barrington ◽

James Briscoe

Keyword(s):

Gene Expression ◽

Spinal Cord ◽

Single Cell ◽

Motor Neurons ◽

Neural Tube ◽

Transcriptome Profiling ◽

Cell Types ◽

Human Embryos ◽

Neuronal Populations ◽

Cell Transcriptome

AbstractThe spinal cord receives input from peripheral sensory neurons and controls motor output by regulating muscle innervating motor neurons. These functions are carried out by neural circuits comprising molecularly and physiologically distinct neuronal subtypes that are generated in a characteristic spatial-temporal arrangement from progenitors in the embryonic neural tube. The systematic mapping of gene expression in mouse embryos has provided insight into the diversity and complexity of cells in the neural tube. For human embryos, however, less information has been available. To address this, we used single cell mRNA sequencing to profile cervical and thoracic regions in four human embryos of Carnegie Stages (CS) CS12, CS14, CS17 and CS19 from Gestational Weeks (W) 4-7. In total we recovered the transcriptomes of 71,219 cells. Analysis of progenitor and neuronal populations from the neural tube, as well as cells of the peripheral nervous system, in dorsal root ganglia adjacent to the neural tube, identified dozens of distinct cell types and facilitated the reconstruction of the differentiation pathways of specific neuronal subtypes. Comparison with existing mouse datasets revealed the overall similarity of mouse and human neural tube development while highlighting specific features that differed between species. These data provide a catalogue of gene expression and cell type identity in the developing neural tube that will support future studies of sensory and motor control systems and can be explored at https://shiny.crick.ac.uk/scviewer/neuraltube/.

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Reversible programming of pluripotent cell differentiation

Journal of Cell Science ◽

10.1242/jcs.113.3.555 ◽

2000 ◽

Vol 113 (3) ◽

pp. 555-566 ◽

Cited By ~ 9

Author(s):

J. Lake ◽

J. Rathjen ◽

J. Remiszewski ◽

P.D. Rathjen

Keyword(s):

Gene Expression ◽

Embryoid Bodies ◽

Visceral Endoderm ◽

Es Cell ◽

Primitive Endoderm ◽

Pluripotent Cell ◽

Pluripotent Cells ◽

Differentiation Potential

We have undertaken an in vitro differentiation analysis of two related, interconvertible, pluripotent cell populations, ES and early primitive ectoderm-like (EPL) cells, which are most similar in morphology, gene expression, cytokine responsiveness and differentiation potential in vivo to ICM and early primitive ectoderm, respectively. Pluripotent cells were differentiated in vitro as aggregates (embryoid bodies) and the appearance and abundance of cell lineages were assessed by morphology and gene expression. Differentiation in EPL cell embryoid bodies recapitulated normal developmental progression in vivo, but was advanced in comparison to ES cell embryoid bodies, with the rapid establishment of late primitive ectoderm specific gene expression, and subsequent loss of pluripotent cell markers. Nascent mesoderm was formed earlier and more extensively in EPL cell embryoid bodies, and resulted in the appearance of terminally differentiated mesodermal cell types prior to and at higher levels than in ES cell embryoid bodies. Nascent mesoderm in EPL cell embryoid bodies was not specified but could be programmed to alternative fates by the addition of exogenous factors. EPL cells remained competent to form primitive endoderm even though this is not the normal fate of primitive ectoderm in vivo. The establishment of primitive ectoderm-like gene expression and inability to participate in embryogenesis following blastocyst injection is therefore not directly associated with restriction in the ability to form extra-embryonic lineages. However, the EPL cell embryoid body environment did not support differentiation of primitive endoderm to visceral endoderm, indicating the lack of an inductive signal for visceral endoderm formation deduced to originate from the pluripotent cells. Similarly, the inability of EPL cells to form neurons when differentiated as embryoid bodies was attributable to perturbation of the differentiation environment and loss of inductive signals rather than a restricted differentiation potential. Reversion of EPL cells to ES cells was accompanied by restoration of ES cell-like differentiation potential. These results demonstrate the ability of pluripotent cells to adopt developmentally distinct, stable cell states with altered differentiation potentials.

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The p75NTR neurotrophin receptor is required to organize the mature neuromuscular synapse by regulating synaptic vesicle availability

Acta Neuropathologica Communications ◽

10.1186/s40478-019-0802-7 ◽

2019 ◽

Vol 7 (1) ◽

Cited By ~ 2

Author(s):

Viviana Pérez ◽

Francisca Bermedo-Garcia ◽

Diego Zelada ◽

Felipe A. Court ◽

Miguel Ángel Pérez ◽

...

Keyword(s):

Neuromuscular Junction ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Neuronal Damage ◽

Neuromuscular Synapse ◽

Force Production ◽

Synaptic Function ◽

Neurotrophin Receptor ◽

Acetylcholinesterase Inhibition ◽

Pharmacological Intervention

Abstract The coordinated movement of organisms relies on efficient nerve-muscle communication at the neuromuscular junction. After peripheral nerve injury or neurodegeneration, motor neurons and Schwann cells increase the expression of the p75NTR pan-neurotrophin receptor. Even though p75NTR targeting has emerged as a promising therapeutic strategy to delay peripheral neuronal damage progression, the effects of long-term p75NTR inhibition at the mature neuromuscular junction have not been elucidated. We performed quantitative neuroanathomical analyses of the neuromuscular junction in p75NTR null mice by laser confocal and electron microscopy, which were complemented with electromyography, locomotor tests, and pharmacological intervention studies. Mature neuromuscular synapses of p75NTR null mice show impaired postsynaptic organization and ultrastructural complexity, which correlate with altered synaptic function at the levels of nerve activity-induced muscle responses, muscle fiber structure, force production, and locomotor performance. Our results on primary myotubes and denervated muscles indicate that muscle-derived p75NTR does not play a major role on postsynaptic organization. In turn, motor axon terminals of p75NTR null mice display a strong reduction in the number of synaptic vesicles and active zones. According to the observed pre and postsynaptic defects, pharmacological acetylcholinesterase inhibition rescued nerve-dependent muscle response and force production in p75NTR null mice. Our findings revealing that p75NTR is required to organize mature neuromuscular junctions contribute to a comprehensive view of the possible effects caused by therapeutic attempts to target p75NTR.

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A Study of Gene Expression Changes in Human Spinal and Oculomotor Neurons; Identifying Potential Links to Sporadic ALS

Genes ◽

10.3390/genes11040448 ◽

2020 ◽

Vol 11 (4) ◽

pp. 448

Author(s):

Aayan N. Patel ◽

Dennis Mathew

Keyword(s):

Gene Expression ◽

Disease Progression ◽

Motor Neurons ◽

Expression Profiles ◽

Gene Expression Profiles ◽

Tissue Samples ◽

Disease Symptoms ◽

Sporadic Als ◽

Oculomotor Neurons ◽

Als Patients

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that causes compromised function of motor neurons and neuronal death. However, oculomotor neurons are largely spared from disease symptoms. The underlying causes for sporadic ALS as well as for the resistance of oculomotor neurons to disease symptoms remain poorly understood. In this bioinformatic-analysis, we compared the gene expression profiles of spinal and oculomotor tissue samples from control individuals and sporadic ALS patients. We show that the genes GAD2 and GABRE (involved in GABA signaling), and CALB1 (involved in intracellular Ca2+ ion buffering) are downregulated in the spinal tissues of ALS patients, but their endogenous levels are higher in oculomotor tissues relative to the spinal tissues. Our results suggest that the downregulation of these genes and processes in spinal tissues are related to sporadic ALS disease progression and their upregulation in oculomotor neurons confer upon them resistance to ALS symptoms. These results build upon prevailing models of excitotoxicity that are relevant to sporadic ALS disease progression and point out unique opportunities for better understanding the progression of neurodegenerative properties associated with sporadic ALS.

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Therapeutic Strategies for Duchenne Muscular Dystrophy: An Update

Genes ◽

10.3390/genes11080837 ◽

2020 ◽

Vol 11 (8) ◽

pp. 837 ◽

Cited By ~ 4

Author(s):

Chengmei Sun ◽

Luoan Shen ◽

Zheng Zhang ◽

Xin Xie

Keyword(s):

Duchenne Muscular Dystrophy ◽

Muscular Dystrophy ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Neuromuscular Disorders ◽

Dystrophin Gene ◽

Current Status ◽

Muscle Stem Cells ◽

Cell Based Therapy ◽

Dystrophin Protein

Neuromuscular disorders encompass a heterogeneous group of conditions that impair the function of muscles, motor neurons, peripheral nerves, and neuromuscular junctions. Being the most common and most severe type of muscular dystrophy, Duchenne muscular dystrophy (DMD), is caused by mutations in the X-linked dystrophin gene. Loss of dystrophin protein leads to recurrent myofiber damage, chronic inflammation, progressive fibrosis, and dysfunction of muscle stem cells. Over the last few years, there has been considerable development of diagnosis and therapeutics for DMD, but current treatments do not cure the disease. Here, we review the current status of DMD pathogenesis and therapy, focusing on mutational spectrum, diagnosis tools, clinical trials, and therapeutic approaches including dystrophin restoration, gene therapy, and myogenic cell transplantation. Furthermore, we present the clinical potential of advanced strategies combining gene editing, cell-based therapy with tissue engineering for the treatment of muscular dystrophy.

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Multiple Presynaptic and Postsynaptic Sites of Inhibitory Modulation by Myomodulin at ARC Neuromuscular Junctions ofAplysia

Journal of Neurophysiology ◽

10.1152/jn.00140.2002 ◽

2003 ◽

Vol 89 (3) ◽

pp. 1488-1502 ◽

Cited By ~ 9

Author(s):

Irina V. Orekhova ◽

Vera Alexeeva ◽

Paul J. Church ◽

Klaudiusz R. Weiss ◽

Vladimir Brezina

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Neuromuscular Junctions ◽

Definitive Evidence ◽

Multiple Parameters ◽

Intact Muscle ◽

Single Arc ◽

Excitatory Junction Potentials ◽

K Current ◽

Neuron Terminals

The functional activity of even simple cellular ensembles is often controlled by surprisingly complex networks of neuromodulators. One such network has been extensively studied in the accessory radula closer (ARC) neuromuscular system of Aplysia. The ARC muscle is innervated by two motor neurons, B15 and B16, which release modulatory peptide cotransmitters to shape ACh-mediated contractions of the muscle. Previous analysis has shown that key to the combinatorial ability of B15 and B16 to control multiple parameters of the contraction is an asymmetry in their peptide modulatory actions. B16, but not B15, releases myomodulin, which, among other actions, inhibits the contraction. Work in single ARC muscle fibers has identified a distinctive myomodulin-activated K current as a candidate postsynaptic mechanism of the inhibition. However, definitive evidence for this mechanism has been lacking. Here, working with the single fibers and then motor neuron-elicited excitatory junction potentials (EJPs) and contractions of the intact ARC muscle, we have confirmed two central predictions of the K-current hypothesis: the myomodulin inhibition of contraction is associated with a correspondingly large inhibition of the underlying depolarization, and the inhibition of both contraction and depolarization is blocked by 4-aminopyridine (4-AP), a potent and selective blocker of the myomodulin-activated K current. However, in the intact muscle, the experiments revealed a second, 4-AP-resistant component of myomodulin inhibition of both B15- and B16-elicited EJPs. This component resembles, and mutually occludes with, inhibition of the EJPs by another peptide modulator released from both B15 and B16, buccalin, which acts by a presynaptic mechanism, inhibition of ACh release from the motor neuron terminals. Direct measurements of peptide release showed that myomodulin also inhibits buccalin release from B15 terminals. At the level of contractions, nevertheless, the postsynaptic K-current mechanism is responsible for much of the myomodulin inhibition of peak contraction amplitude. The presynaptic mechanism, which is most evident during the initial build-up of the EJP waveform, underlies instead an increase of contraction latency.

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