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

Cristina Pinto,Viviana Pérez,Miguel Albistur,Juan Pablo Henríquez,Jessica Mella,Teresa Caprile,Francisca C Bronfman

doi:10.3390/biom11121898

Cristina Pinto, Viviana Pérez + Show 5 more

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https://doi.org/10.3390/biom11121898

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Abstract

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.

Highlights

The vertebrate neuromuscular junction (NMJ) allows muscle contraction and controls the coordinated movement of organisms
Even though double staining was not possible due to technical limitations, in adjacent chick embryo sections we found that the endogenous synaptic vesicle 2 protein (SV2) follows a comparable distribution and puncta size to cWnt3HA staining (Figure 2F)
We considered that the Drosophila Wnt orthologue Wingless (Wg) is secreted in exosomes associated with the Wnt-binding protein Evenness Interrupted (Evi) at the fly NMJ, via a mechanism dependent on Rab11 and Syntaxin1A (Syx1A) [27,28]

Summary

Introduction

The vertebrate neuromuscular junction (NMJ) allows muscle contraction and controls the coordinated movement of organisms. During embryonic NMJ assembly, motor axons that invade nascent muscle fibers undergo presynaptic differentiation and accumulate synaptic vesicles containing acetylcholine and trophic factors. NMJ assembly relies on trans-synaptic signaling triggered by secreted and extracellular matrix molecules from motor axons, skeletal muscle fibers, and tSCs [1,2]. The motor neuron-derived proteoglycan agrin plays major roles on the assembly and maintenance of postsynaptic acetylcholine receptors on the muscle membrane [3]. In this regard, cumulative evidence reveals that Wnt signaling pathways control crucial steps of NMJ synaptogenesis

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