Transport and Secretion of the Wnt3 Ligand by Motor Neuron-like Cells and Developing Motor Neurons

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.

Download Full-text

Interaction of 125I-labeled botulinum neurotoxins with nerve terminals. I. Ultrastructural autoradiographic localization and quantitation of distinct membrane acceptors for types A and B on motor nerves.

The Journal of Cell Biology ◽

10.1083/jcb.103.2.521 ◽

1986 ◽

Vol 103 (2) ◽

pp. 521-534 ◽

Cited By ~ 174

Author(s):

J D Black ◽

J O Dolly

Keyword(s):

Plasma Membrane ◽

Neuromuscular Junction ◽

Nerve Terminal ◽

Motor Nerve ◽

Type A ◽

Nerve Trunk ◽

Metabolic Inhibitors ◽

Motor Nerve Terminal ◽

Electron Microscopic Autoradiography

The labeling patterns produced by radioiodinated botulinum neurotoxin (125I-BoNT) types A and B at the vertebrate neuromuscular junction were investigated using electron microscopic autoradiography. The data obtained allow the following conclusions to be made. 125I-BoNT type A, applied in vivo or in vitro to mouse diaphragm or frog cutaneous pectoris muscle, interacts saturably with the motor nerve terminal only; silver grains occur on the plasma membrane, within the synaptic bouton, and in the axoplasm of the nerve trunk, suggesting internalization and retrograde intra-axonal transport of toxin or fragments thereof. 125I-BoNT type B, applied in vitro to the murine neuromuscular junction, interacts likewise with the motor nerve terminal except that a lower proportion of internalized radioactivity is seen. This result is reconcilable with the similar, but not identical, pharmacological action of these toxin types. The saturability of labeling in each case suggested the involvement of acceptors; on preventing the internalization step with metabolic inhibitors, their precise location became apparent. They were found on all unmyelinated areas of the nerve terminal membrane, including the preterminal axon and the synaptic bouton. Although 125I-BoNT type A interacts specifically with developing terminals of newborn rats, the unmyelinated plasma membrane of the nerve trunk is not labeled, indicating that the acceptors are unique components restricted to the nerve terminal area. BoNT types A and B have distinct acceptors on the terminal membrane. Having optimized the conditions for saturation of these binding sites and calibrated the autoradiographic procedure, we found the densities of the acceptors for types A and B to be approximately 150 and 630/micron 2 of membrane, respectively. It is proposed that these membrane acceptors target BoNT to the nerve terminal and mediate its delivery to an intracellular site, thus contributing to the toxin's selective inhibitory action on neurotransmitter release.

Download Full-text

Increased Transmitter Release and Aberrant Synapse Morphology in a Drosophila Calmodulin Mutant

Genetics ◽

10.1093/genetics/150.1.265 ◽

1998 ◽

Vol 150 (1) ◽

pp. 265-274 ◽

Cited By ~ 1

Author(s):

LaChelle Arredondo ◽

Heidi B Nelson ◽

Kathy Beckingham ◽

Michael Stern

Keyword(s):

Nerve Terminal ◽

Calcium Binding ◽

Motor Nerve ◽

K Channel ◽

Motor Nerve Terminal ◽

Larval Neuromuscular Junction ◽

Eukaryotic Organisms ◽

And Function ◽

Calmodulin Mutant

Abstract The ubiquitous calcium-binding protein calmodulin (CaM) has been implicated in the development and function of the nervous system in a variety of eukaryotic organisms. We have generated mutations in the single Drosophila Calmodulin (Cam) gene and examined the effects of these mutations on behavior, synaptic transmission at the larval neuromuscular junction, and structure of the larval motor nerve terminal. Flies hemizygous for Cam3c1, a mutation in the first Ca2+-binding site, exhibit behavioral, neurophysiological, and neuroanatomical abnormalities. In particular, adults exhibit defects in locomotion, coordination, and flight. Larvae exhibit increased neurotransmitter release from the motor nerve terminal at low [Ca2+] in the presence of the K+ channel-blocking drug quinidine. In addition, synaptic bouton structure at motor nerve terminals is altered. These effects are distinct from those produced by altering the activity of the CaM target enzymes CaM-activated kinase II (CaMKII) and CaM-activated adenylyl cyclase (CaMAC). Furthermore, previous in vitro studies of mutant Cam3c1 demonstrated that although its Ca2+ affinity is decreased, Cam3c1 protein can activate CaMKII, CaMAC, and CaM-activated phosphatase calcineurin in a manner similar to wild-type CaM. Thus, the Cam3c1 mutation might affect Ca2+ buffering or interfere with the activation or inhibition of a CaM target distinct from CaMKII or CaMAC.

Download Full-text

“Stretch-Growth” of Motor Axons in Custom Mechanobioreactors to Generate Long-Projecting Axonal and Axonal-Myocyte Constructs

10.1101/598755 ◽

2019 ◽

Cited By ~ 1

Author(s):

Kritika S. Katiyar ◽

Laura A. Struzyna ◽

Suradip Das ◽

D. Kacy Cullen

Keyword(s):

Spinal Cord ◽

Nervous System ◽

Motor Neuron ◽

Motor Neurons ◽

Motor Axon ◽

Central Feature ◽

Motor Axons ◽

Ganglion Neurons

AbstractThe central feature of peripheral motor axons is their remarkable lengths as they project from a motor neuron residing in the spinal cord to an often-distant target muscle. However, to date in vitro models have not replicated this central feature owing to challenges in generating motor axon tracts beyond a few millimeters in length. To address this, we have developed a novel combination of micro-tissue engineering and mechanically assisted growth techniques to create long-projecting centimeter-scale motor axon tracts. Here, primary motor neurons were isolated from the spinal cords of rats and induced to form engineered micro-spheres via forced aggregation in custom micro-wells. This three-dimensional micro-tissue yielded healthy motor neurons projecting dense, fasciculated axonal tracts. Within our custom-built mechanobioreactors, motor neuron culture conditions, neuronal/axonal architecture, and mechanical growth conditions were systematically optimized to generate parameters for robust and efficient “stretch-growth” of motor axons. We found that axons projecting from motor neuron aggregates were able to respond to axon displacement rates at least 10 times greater than that tolerated by axons projecting from dissociated motor neurons. The growth and structural characteristics of these stretch-grown motor axons were compared to benchmark stretch-grown axons from sensory dorsal root ganglion neurons, revealing similar axon densities yet increased motor axon fasciculation. Finally, motor axons were integrated with myocytes and then stretch-grown to create novel long-projecting axonal-myocyte constructs that better recreate characteristic dimensions of native nerve-muscle anatomy. This is the first demonstration of mechanical elongation of spinal cord motor axons and may have applications as anatomically inspired in vitro testbeds or as tissue engineered “living scaffolds” for targeted axon tract reconstruction following nervous system injury or disease.Significance StatementWe have developed novel axon tracts of unprecedented lengths spanning either two discrete populations of neurons or a population of neurons and skeletal myocytes. This is the first demonstration of “stretch-grown” motor axons that recapitulate the structure of spinal motor neurons in vivo by projecting long axons from a pool of motor neurons to distant targets, and may have applications as anatomically inspired in vitro test beds to study mechanisms of axon growth, development, and neuromuscular function in anatomically accurate axo-myo constructs; as well as serve as “living scaffolds” in vivo for targeted axon tract reconstruction following nervous system trauma.

Download Full-text

Modeling motor neuron resilience in ALS using stem cells

10.1101/399659 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ilary Allodi ◽

Jik Nijssen ◽

Julio Aguila Benitez ◽

Christoph Schweingruber ◽

Andrea Fuchs ◽

...

Keyword(s):

Stem Cells ◽

Motor Neuron ◽

Motor Neurons ◽

Embryonic Stem ◽

Spinal Motor Neurons ◽

Neuronal Progenitors ◽

Oculomotor Neurons ◽

Bona Fide

SUMMARYOculomotor neurons, which regulate eye movement, are resilient to degeneration in the lethal motor neuron disease amyotrophic lateral sclerosis (ALS). It would be highly advantageous if motor neuron resilience could be modeled in vitro. Towards this goal, we generated a high proportion of oculomotor neurons from mouse embryonic stem cells through temporal overexpression of Phox2a in neuronal progenitors. We demonstrate, using electrophysiology, immunocytochemistry and RNA sequencing, that in vitro generated neurons are bona fide oculomotor neurons based on their cellular properties and similarity to their in vivo counterpart in rodent and man. We also show that in vitro generated oculomotor neurons display a robust activation of survival-promoting Akt signaling and are more resilient to the ALS-like toxicity of kainic acid than spinal motor neurons. Thus, we can generate bona fide oculomotor neurons in vitro which display a resilience similar to that seen in vivo.

Download Full-text

In Vivo Single-Molecule Tracking at the Drosophila Presynaptic Motor Nerve Terminal

Journal of Visualized Experiments ◽

10.3791/56952 ◽

2018 ◽

Cited By ~ 3

Author(s):

Adekunle T. Bademosi ◽

Elsa Lauwers ◽

Rumelo Amor ◽

Patrik Verstreken ◽

Bruno van Swinderen ◽

...

Keyword(s):

Single Molecule ◽

Nerve Terminal ◽

Motor Nerve ◽

Motor Nerve Terminal ◽

Single Molecule Tracking

Download Full-text

Clinical Safety and Tolerability of A2NTX, a Novel Low Molecular Weight Neurotoxin Derived From Botulinum Toxin Subtype A2, in Comparison with Subtype A1 Toxins

10.20944/preprints202110.0121.v1 ◽

2021 ◽

Author(s):

Ryuji Kaji ◽

Toshiaki Takeuchi ◽

Takefumi Okuno ◽

Ai Miyashiro ◽

Tomoko Kohda ◽

...

Keyword(s):

Molecular Weight ◽

Open Study ◽

Motor Nerve ◽

Tolerability Profile ◽

Low Molecular Weight ◽

Motor Nerve Terminal ◽

Clinical Safety ◽

Botulinum Type

All the available botulinum type A neurotoxins for clinical uses are of A1 subtype. We developed a subtype A2 low molecular weight (150kD) neurotoxin (A2NTX), with less spread and faster entry into the motor nerve terminal than A1 in vitro and in vivo. Preliminary clinical studies showed its efficacy superior to A1 toxins. We conducted an open study exploring its safety and tolerability profile in comparison with A1LL (onabotulinumtoxinA) and low molecular weight (150kD) A1 neurotoxin (A1NTX). Those who had been using A1LL (n=90; 50-360 mouse LD50 units) or A1NTX (n=30; 50-580 units) were switched to A2NTX (n=120; 25-600 units) from 2010 till 2018 (number of sessions ~ 27, cumulative doses ~11,640 units per patient). Adverse events for A2NTX included weakness (n=1, ascribed to alcoholic polyneuropathy), dysphagia (1), local weakness (4), spread to other muscles (1), whereas those for A1LL or A1NTX comprised weakness (n=2, A1NTX), dysphagia (8), ptosis (6), local weakness (7) and spread to other muscles (15). After injections, 89 out of 120 patients preferred A2NTX to A1 for the successive sessions. The present study demonstrated that A2NTX had the clinical safety up to the dose of 500 units, and was well tolerated compared to A1 toxins.

Download Full-text

Astrocytic expression of ALS-causative mutant FUS leads to TNFa-dependent neurodegeneration in vivo

10.1101/2021.11.23.469650 ◽

2021 ◽

Author(s):

Brigid K Jensen ◽

Kevin J McAvoy ◽

Nicolette M Heinsinger ◽

Angelo C Lepore ◽

Hristelina Ilieva ◽

...

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Neutralizing Antibodies ◽

Rna Binding ◽

Neuronal Damage ◽

Specific Expression ◽

Fused In Sarcoma ◽

Knockout Animals

Genetic mutations that cause Amyotrophic Lateral Sclerosis (ALS), a progressively lethal motor neuron disease, are commonly found in ubiquitously expressed genes. In addition to direct defects within motor neurons, growing evidence suggests that dysfunction of non-neuronal cells is also an important driver of disease. Previously, we demonstrated that mutations in DNA/RNA binding protein Fused in Sarcoma (FUS) induce neurotoxic phenotypes in astrocytes in vitro, via activation of the NF-κB pathway and release of pro-inflammatory cytokine TNFα. Here, we developed an intraspinal cord injection model to test whether astrocyte-specific expression of ALS-causative FUSR521G variant (mtFUS) causes neuronal damage in vivo. We show that mtFUS expression causes TNFα upregulation, motor function deficits, and spinal motor neuron loss. We further demonstrate a lack of phenotype in TNFα knockout animals expressing mtFUS, and prevention of neurodegeneration in mtFUS-transduced animals through administration of TNFα neutralizing antibodies. Together, these studies strengthen evidence that astrocytes contribute to disease in ALS, establish that FUS-ALS astrocytes induce pathogenic changes to motor neurons in vivo, and provide insights identifying FUS-ALS specific potential therapeutic targets.

Download Full-text

Presynaptic Proteins as Markers of the Neurotoxic Activity of BmjeTX-I and BmjeTX-II Toxins fromBothrops marajoensis(Marajó Lancehead) Snake Venom

Biochemistry Research International ◽

10.1155/2016/2053459 ◽

2016 ◽

Vol 2016 ◽

pp. 1-10

Author(s):

Antonio Lisboa ◽

Rodolfo Melaré ◽

Junia R. B. Franco ◽

Carolina V. Bis ◽

Marta Gracia ◽

...

Keyword(s):

Nerve Terminal ◽

Neurotransmitter Release ◽

Acetylcholine Receptors ◽

Motor Nerve ◽

Synaptic Proteins ◽

Muscle Fibres ◽

Motor Nerve Terminal ◽

Transmission Electron ◽

Skeletal Muscle Fibres

Neuromuscular preparations exposed toB. marajoensisvenom show increases in the frequency of miniature end-plate potentials and twitch tension facilitation followed by presynaptic neuromuscular paralysis, without evidences of muscle damage. Considering that presynaptic toxins interfere into the machinery involved in neurotransmitter release (synaptophysin, synaptobrevin, and SNAP25 proteins), the main objective of this communication is to analyze, by immunofluorescence and western blotting, the expression of the synaptic proteins, synaptophysin, synaptobrevin, and SNAP25 and by myography, light, and transmission electron microscopy the pathology of motor nerve terminals and skeletal muscle fibres of chick biventer cervicis preparations (CBC) exposedin vitroto BmjeTX-I and BmjeTX-II toxins fromB. marajoensisvenom. CBC incubated with toxins showed irreversible twitch tension blockade and unaffected KCl- and ACh-evoked contractures, and the positive colabelling of acetylcholine receptors confirmed that their action was primarily at the motor nerve terminal. Hypercontraction and loose myofilaments and synaptic vesicle depletion and motor nerve damage indicated that the toxins displayed both myotoxic and neurotoxic effect. The blockade resulted from interference on synaptophysin, synaptobrevin, and SNAP25 proteins leading to the conclusion that BmjeTX-I and BmjeTX-II affected neurotransmitter release machinery by preventing the docking of synaptic vesicles to the axolemma of the nerve terminal.

Download Full-text

Neonatal Death in Mice Lacking Cardiotrophin-like Cytokine is Associated with Multifocal Neuronal Hypoplasia

Veterinary Pathology ◽

10.1354/vp.08-vp-0239-b-bc ◽

2008 ◽

Vol 46 (3) ◽

pp. 514-519 ◽

Cited By ~ 18

Author(s):

X. Zou ◽

B. Bolon ◽

J. K. Pretorius ◽

C. Kurahara ◽

J. McCabe ◽

...

Keyword(s):

Motor Neuron ◽

Motor Neurons ◽

Ventral Horn ◽

Lumbar Spinal Cord ◽

Facial Nucleus ◽

In Ovo ◽

Neuron Survival ◽

Motor Neuron Survival ◽

Lumbar Spinal

Mice with null mutations of ciliary neurotrophic factor (Cntf) receptor alpha (Cntf-Rα), or cytokine-like factor 1 (Clf), one component of Cntf-II (a heterodimeric Cntf-Rα ligand), die as neonates from motor neuron loss affecting the facial nucleus and ventral horn of the lumbar spinal cord. Exposure to cardiotrophin-like cytokine (Clc), the other putative Cntf-II element, supports motor neuron survival in vitro and in ovo. Confirmation that Clc ablation induces an equivalent phenotype to Clf deletion would support a role for Clc in the functional Cntf-II complex. In this study, Clc knockout mice had decreased facial motility, did not suckle, died within 24 hours, and had 32% and 29% fewer motor neurons in the facial nucleus and lumbar ventral horn, respectively; thus, Clc is essential for motor neuron survival during development. The concordance of the Clc knockout phenotype with those of mice lacking Cntf-Rα or Clf bolsters the hypothesis that Clc participates in Cntf-II.

Download Full-text

A motor neuron disease–associated mutation in p150Glued perturbs dynactin function and induces protein aggregation

The Journal of Cell Biology ◽

10.1083/jcb.200511068 ◽

2006 ◽

Vol 172 (5) ◽

pp. 733-745 ◽

Cited By ~ 126

Author(s):

Jennifer R. Levy ◽

Charlotte J. Sumner ◽

Juliane P. Caviston ◽

Mariko K. Tokito ◽

Srikanth Ranganathan ◽

...

Keyword(s):

Cell Death ◽

Motor Neuron ◽

Motor Neurons ◽

Mitotic Spindle ◽

Cytoplasmic Dynein ◽

Aggregate Formation ◽

Microtubule Motor ◽

Delayed Recovery

The microtubule motor cytoplasmic dynein and its activator dynactin drive vesicular transport and mitotic spindle organization. Dynactin is ubiquitously expressed in eukaryotes, but a G59S mutation in the p150Glued subunit of dynactin results in the specific degeneration of motor neurons. This mutation in the conserved cytoskeleton-associated protein, glycine-rich (CAP-Gly) domain lowers the affinity of p150Glued for microtubules and EB1. Cell lines from patients are morphologically normal but show delayed recovery after nocodazole treatment, consistent with a subtle disruption of dynein/dynactin function. The G59S mutation disrupts the folding of the CAP-Gly domain, resulting in aggregation of the p150Glued protein both in vitro and in vivo, which is accompanied by an increase in cell death in a motor neuron cell line. Overexpression of the chaperone Hsp70 inhibits aggregate formation and prevents cell death. These data support a model in which a point mutation in p150Glued causes both loss of dynein/dynactin function and gain of toxic function, which together lead to motor neuron cell death.

Download Full-text