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Stem Cell Research Oct 2018Primary rodent neurons and immortalised cell lines have overwhelmingly been used for in vitro studies of traumatic injury to peripheral and central neurons, but have...
Primary rodent neurons and immortalised cell lines have overwhelmingly been used for in vitro studies of traumatic injury to peripheral and central neurons, but have some limitations of physiological accuracy. Motor neurons (MN) derived from human induced pluripotent stem cells (iPSCs) enable the generation of cell models with features relevant to human physiology. To facilitate this, it is desirable that MN protocols both rapidly and efficiently differentiate human iPSCs into electrophysiologically active MNs. In this study, we present a simple, rapid protocol for differentiation of human iPSCs into functional spinal (lower) MNs, involving only adherent culture and use of small molecules for directed differentiation, with the ultimate aim of rapid production of electrophysiologically functional cells for short-term neural injury experiments. We show successful differentiation in two unrelated iPSC lines, by quantifying neural-specific marker expression, and by evaluating cell functionality at different maturation stages by calcium imaging and patch clamping. Differentiated neurons were shown to be electrophysiologically altered by uniaxial mechanical deformation. Spontaneous network activity decreased with applied stretch, indicating aberrant network connectivity. These results demonstrate the feasibility of this rapid, simple protocol for differentiating iPSC-derived MNs, suitable for in vitro neural injury studies focussing on electrophysiological alterations caused by mechanical deformation or trauma.
Topics: Cell Differentiation; Cells, Cultured; Electrophysiology; Humans; Induced Pluripotent Stem Cells; Motor Neurons
PubMed: 30278374
DOI: 10.1016/j.scr.2018.09.006 -
The Neuroscientist : a Review Journal... Feb 2009Neurotrophic factors (NTFs) are a pleiotropic group of secreted growth factors that regulate multiple aspects of neuronal development, including the regressive event of... (Review)
Review
Neurotrophic factors (NTFs) are a pleiotropic group of secreted growth factors that regulate multiple aspects of neuronal development, including the regressive event of cell death. Skeletal muscleinnervating lower motoneurons (MNs) of the brain stem and spinal cord comprise one population of central neurons in which programmed cell death (PCD) during embryogenesis has been actively investigated, as much for reasons of technical facility as clinical relevance. The precise identity of NTF-dependent MNs has remained unclear, with most studies simply reporting losses or gains across the entire spinal cord or individual brain-stem nuclei. However, MNs are grouped into highly heterogenous populations based on transcriptional identity, target innervation, and physiological function. Therefore, recent work has focused on the effects of NTF overexpression or deletion on the survival of these MN subpopulations. Together with the recent progress attained in the generation of conditional mutant mice, in which the function of an NTF or its receptor can be eliminated specifically in MNs, these recent studies have begun to define the differential trophic requirements for MN subpopulations during PCD. The intent of this review is to summarize these recent findings and to discuss their significance with respect to neurotrophic theory.
Topics: Animals; Motor Neurons; Nerve Growth Factors
PubMed: 19218234
DOI: 10.1177/1073858408324787 -
The Journal of Physiology Apr 2018Although adenosine 2A (A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing...
KEY POINTS
Although adenosine 2A (A ) receptor activation triggers specific cell signalling cascades, the ensuing physiological outcomes depend on the specific cell type expressing these receptors. Cervical spinal adenosine 2A (A ) receptor activation elicits a prolonged facilitation in phrenic nerve activity, which was nearly abolished following intrapleural A receptor siRNA injections. A receptor siRNA injections selectively knocked down A receptors in cholera toxin B-subunit-identified phrenic motor neurons, sparing cervical non-phrenic motor neurons. Collectively, our results support the hypothesis that phrenic motor neurons express the A receptors relevant to A receptor-induced phrenic motor facilitation. Upregulation of A receptor expression in the phrenic motor neurons per se may potentially be a useful approach to increase phrenic motor neuron excitability in conditions such as spinal cord injury.
ABSTRACT
Cervical spinal adenosine 2A (A ) receptor activation elicits a prolonged increase in phrenic nerve activity, an effect known as phrenic motor facilitation (pMF). The specific cervical spinal cells expressing the relevant A receptors for pMF are unknown. This is an important question since the physiological outcome of A receptor activation is highly cell type specific. Thus, we tested the hypothesis that the relevant A receptors for pMF are expressed in phrenic motor neurons per se versus non-phrenic neurons of the cervical spinal cord. A receptor immunostaining significantly colocalized with NeuN-positive neurons (89 ± 2%). Intrapleural siRNA injections were used to selectively knock down A receptors in cholera toxin B-subunit-labelled phrenic motor neurons. A receptor knock-down was verified by a ∼45% decrease in A receptor immunoreactivity within phrenic motor neurons versus non-targeting siRNAs (siNT; P < 0.05). There was no evidence for knock-down in cervical non-phrenic motor neurons. In rats that were anaesthetized, subjected to neuromuscular blockade and ventilated, pMF induced by cervical (C3-4) intrathecal injections of the A receptor agonist CGS21680 was greatly attenuated in siA (21%) versus siNT treated rats (147%; P < 0.01). There were no significant effects of siA on phrenic burst frequency. Collectively, our results support the hypothesis that phrenic motor neurons express the A receptors relevant to A receptor-induced pMF.
Topics: Action Potentials; Adenosine A2 Receptor Agonists; Animals; Cholera Toxin; Male; Motor Neurons; Phrenic Nerve; Rats; Rats, Sprague-Dawley; Receptor, Adenosine A2A
PubMed: 29388230
DOI: 10.1113/JP275462 -
Cell Biochemistry and Function Jan 2020Desflurane is one of the commonly used general anaesthetics. Recently, it was reported that desflurane caused neurotoxicity, raising concerns in clinical use. In this...
Desflurane is one of the commonly used general anaesthetics. Recently, it was reported that desflurane caused neurotoxicity, raising concerns in clinical use. In this study, we found desflurane could affect viability and maturation in motor neurons. Dexmedetomidine, a α2-adrenergic receptor agonist, could attenuate the effect of desflurane on motor neurons. This process was mediated by NF-KappaB signalling. Interestingly, we also found that dexmedetomidine could recover the lesion in motor function and memory impaired by desflurane. Collectively, our results showed the neurotoxic effect of desflurane in motor neurons. More importantly, this process was alleviated by dexmedetomidine, potentially showing its application in protecting motor neuron from neurotoxic agents. Significance of the study: This work provides the evidence to support the protective role of dexmedetomidine in desflurane-induced motor neuron death. Since desflurane is a widely used anaesthetic in surgery and leads to neuron death, the neuroprotective effect of dexmedetomidine holds promising clinical application.
Topics: Animals; Cell Death; Cells, Cultured; Desflurane; Dexmedetomidine; Dose-Response Relationship, Drug; Maze Learning; Mice; Motor Neurons; NF-kappa B; Signal Transduction; Structure-Activity Relationship
PubMed: 31774572
DOI: 10.1002/cbf.3439 -
Biochemical Society Transactions Dec 2013MNDs (motor neuron diseases) form a heterogeneous group of pathologies characterized by the progressive degeneration of motor neurons. More and more genetic factors... (Review)
Review
MNDs (motor neuron diseases) form a heterogeneous group of pathologies characterized by the progressive degeneration of motor neurons. More and more genetic factors associated with MND encode proteins that have a function in RNA metabolism, suggesting that disturbed RNA metabolism could be a common underlying problem in several, perhaps all, forms of MND. In the present paper we review recent developments showing a functional link between SMN (survival of motor neuron), the causative factor of SMA (spinal muscular atrophy), and FUS (fused in sarcoma), a genetic factor in ALS (amyotrophic lateral sclerosis). SMN is long known to have a crucial role in the biogenesis and localization of the spliceosomal snRNPs (small nuclear ribonucleoproteins), which are essential assembly modules of the splicing machinery. Now we know that FUS interacts with SMN and pathogenic FUS mutations have a significant effect on snRNP localization. Together with other recently published evidence, this finding potentially links ALS pathogenesis to disturbances in the splicing machinery, and implies that pre-mRNA splicing may be the common weak point in MND, although other steps in mRNA metabolism could also play a role. Certainly, further comparison of the RNA metabolism in different MND will greatly help our understanding of the molecular causes of these devastating diseases.
Topics: Amyotrophic Lateral Sclerosis; Animals; Humans; Motor Neurons; Muscular Atrophy, Spinal; RNA, Messenger
PubMed: 24256260
DOI: 10.1042/BST20130142 -
Current Opinion in Neurobiology Aug 2016Localization and local translation of mRNA plays a key role in neuronal development and function. While studies in various systems have provided insights into molecular... (Review)
Review
Localization and local translation of mRNA plays a key role in neuronal development and function. While studies in various systems have provided insights into molecular mechanisms of mRNA transport and local protein synthesis, the factors that control the assembly of mRNAs and mRNA binding proteins into messenger ribonucleoprotein (mRNP) transport granules remain largely unknown. In this review we will discuss how insights on a motor neuron disease, spinal muscular atrophy (SMA), is advancing our understanding of regulated assembly of transport competent mRNPs and how defects in their assembly and delivery may contribute to the degeneration of motor neurons observed in SMA and other neurological disorders.
Topics: Humans; Motor Neurons; Muscular Atrophy, Spinal; Protein Transport; Ribonucleoproteins
PubMed: 27131421
DOI: 10.1016/j.conb.2016.04.004 -
Current Topics in Developmental Biology 2009Motor behaviors are the primary means by which animals interact with their environment, forming the final output of most central nervous system (CNS) activity. The... (Review)
Review
Motor behaviors are the primary means by which animals interact with their environment, forming the final output of most central nervous system (CNS) activity. The neural circuits that govern basic locomotor functions appear to be genetically hard wired and are comprised of discrete groups of neurons residing within the spinal cord. These local microcircuits coordinate simple reflexive behaviors in response to sensory stimuli and underlie the generation of rhythmic patterns of neural activity necessary for walking. In recent years there have been significant advances in understanding the genetic and molecular programs that determine the specificity of neural connections within the spinal cord that are critical for the emergence of coordinate motor behaviors. The assembly of circuits within the spinal cord requires the generation of diverse cell types to accommodate the intricate sets of interconnections between motor neurons, sensory neurons, interneurons, and muscle. The first and most critical aspect of this process is that motor neurons select their appropriate muscle targets in the periphery with fidelity and precision. All of the subsequent steps in motor neuron connectivity, such as their descending inputs from higher brain centers, their circuits with sensory neurons and interneurons are constrained by the early connections formed between motor neurons and their muscle targets. The actions of a single family of transcription factors, encoded by the chromosomally clustered Hox genes, appear to have a central role in defining the specificity of motor neuron-muscle connectivity. The emerging logic of Hox protein function in motor neuron specification may provide more general insights into the programs that determine synaptic specificity in other CNS regions.
Topics: Animals; Gene Expression Regulation, Developmental; Gene Regulatory Networks; Hindlimb; Homeodomain Proteins; Models, Biological; Motor Activity; Motor Neurons; Transcription Factors
PubMed: 19651305
DOI: 10.1016/S0070-2153(09)88006-X -
Development (Cambridge, England) Feb 2014All muscle movements, including breathing, walking, and fine motor skills rely on the function of the spinal motor neuron to transmit signals from the brain to... (Review)
Review
All muscle movements, including breathing, walking, and fine motor skills rely on the function of the spinal motor neuron to transmit signals from the brain to individual muscle groups. Loss of spinal motor neuron function underlies several neurological disorders for which treatment has been hampered by the inability to obtain sufficient quantities of primary motor neurons to perform mechanistic studies or drug screens. Progress towards overcoming this challenge has been achieved through the synthesis of developmental biology paradigms and advances in stem cell and reprogramming technology, which allow the production of motor neurons in vitro. In this Primer, we discuss how the logic of spinal motor neuron development has been applied to allow generation of motor neurons either from pluripotent stem cells by directed differentiation and transcriptional programming, or from somatic cells by direct lineage conversion. Finally, we discuss methods to evaluate the molecular and functional properties of motor neurons generated through each of these techniques.
Topics: Animals; Cell Lineage; Humans; Motor Neurons; Neurogenesis; Pluripotent Stem Cells; Spinal Cord; Transcription, Genetic
PubMed: 24449832
DOI: 10.1242/dev.097410 -
Journal of Neurology Jan 2019Amyotrophic lateral sclerosis (ALS) is the most common neurodegenerative disease affecting motor neurons (MN). This fatal disease is characterized by progressive muscle... (Review)
Review
Amyotrophic lateral sclerosis (ALS) is the most common neurodegenerative disease affecting motor neurons (MN). This fatal disease is characterized by progressive muscle wasting and lacks an effective treatment. ALS pathogenesis has not been elucidated yet. In a small proportion of ALS patients, the disease has a familial origin, related to mutations in specific genes, which directly result in MN degeneration. By contrast, the vast majority of cases are though to be sporadic, in which genes and environment interact leading to disease in genetically predisposed individuals. Lately, the role of the environment has gained relevance in this field and an extensive list of environmental conditions have been postulated to be involved in ALS. Among them, infectious agents, particularly viruses, have been suggested to play an important role in the pathogenesis of the disease. These agents could act by interacting with some crucial pathways in MN degeneration, such as gene processing, oxidative stress or neuroinflammation. In this article, we will review the main studies about the involvement of microorganisms in ALS, subsequently discussing their potential pathogenic effect and integrating them as another piece in the puzzle of ALS pathogenesis.
Topics: Amyotrophic Lateral Sclerosis; Animals; Bacterial Infections; Humans; Motor Neurons; Virus Diseases
PubMed: 29845377
DOI: 10.1007/s00415-018-8919-3 -
Brain : a Journal of Neurology Sep 2011Frontotemporal dementia and motor neuron disease share clinical, genetic and pathological characteristics. Motor neuron disease develops in a proportion of patients with...
Frontotemporal dementia and motor neuron disease share clinical, genetic and pathological characteristics. Motor neuron disease develops in a proportion of patients with frontotemporal dementia, but the incidence, severity and functional significance of motor system dysfunction in patients with frontotemporal dementia has not been determined. Neurophysiological biomarkers have been developed to document motor system dysfunction including: short-interval intracortical inhibition, a marker of corticospinal motor neuron dysfunction and the neurophysiological index, a marker of lower motor neuron dysfunction. The present study performed detailed clinical and neurophysiological assessments on 108 participants including 40 consecutive patients with frontotemporal dementia, 42 age- and gender-matched patients with motor neuron disease and 26 control subjects. Of the 40 patients with frontotemporal dementia, 12.5% had concomitant motor neuron disease. A further 27.3% of the patients with frontotemporal dementia had clinical evidence of minor motor system dysfunction such as occasional fasciculations, mild wasting or weakness. Biomarkers of motor system function were abnormal in frontotemporal dementia. Average short-interval intracortical inhibition was reduced in frontotemporal dementia (4.3 ± 1.7%) compared with controls (9.1 ± 1.1%, P < 0.05). Short-interval intracortical inhibition was particularly reduced in the progressive non-fluent aphasia subgroup, but was normal in patients with behavioural variant frontotemporal dementia and semantic dementia. The neurophysiological index was reduced in frontotemporal dementia (1.1) compared with controls (1.9, P < 0.001), indicating a degree of lower motor neuron dysfunction, although remained relatively preserved when compared with motor neuron disease (0.7, P < 0.05). Motor system dysfunction in frontotemporal dementia may result from pathological involvement of the primary motor cortex, with secondary degeneration of lower motor neurons in the brainstem and anterior horn of the spinal cord.
Topics: Aged; Aged, 80 and over; Biomarkers; Cross-Sectional Studies; Frontotemporal Dementia; Humans; Male; Middle Aged; Motor Neuron Disease; Motor Neurons; Neuropsychological Tests; Transcranial Magnetic Stimulation
PubMed: 21840887
DOI: 10.1093/brain/awr195