Efficient gene transfer into primary muscle cells to analyze nerve-independent postsynaptic organization in vitro

Mella, Jessica; Pérez, Viviana; Zelada, Diego; Moreno, N. E. Martinez; Ionescu, Ariel; Perlson, Eran; Henríquez, Juan Pablo

doi:10.1016/j.nmd.2019.05.005

Cited by 3 publications

(4 citation statements)

References 26 publications

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“…Previous findings have shown that lithium reduces the number of nAChRs in the absence of motor neuron input and in primary muscle cell cultures 36 , 37 but the potential mechanisms involved are unknown. Therefore, we next explored the effect of lithium treatment on postsynaptic stability after LAL muscle denervation inflicted by a 4 mm resection of the facial nerve 38 (Fig.…”

Section: Resultsmentioning

confidence: 97%

Lithium causes differential effects on postsynaptic stability in normal and denervated neuromuscular synapses

Zelada

Barrantes

Henríquez

2021

Sci Rep

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Lithium chloride has been widely used as a therapeutic mood stabilizer. Although cumulative evidence suggests that lithium plays modulatory effects on postsynaptic receptors, the underlying mechanism by which lithium regulates synaptic transmission has not been fully elucidated. In this work, by using the advantageous neuromuscular synapse, we evaluated the effect of lithium on the stability of postsynaptic nicotinic acetylcholine receptors (nAChRs) in vivo. We found that in normally innervated neuromuscular synapses, lithium chloride significantly decreased the turnover of nAChRs by reducing their internalization. A similar response was observed in CHO-K1/A5 cells expressing the adult muscle-type nAChRs. Strikingly, in denervated neuromuscular synapses, lithium led to enhanced nAChR turnover and density by increasing the incorporation of new nAChRs. Lithium also potentiated the formation of unstable nAChR clusters in non-synaptic regions of denervated muscle fibres. We found that denervation-dependent re-expression of the foetal nAChR γ-subunit was not altered by lithium. However, while denervation inhibits the distribution of β-catenin within endplates, lithium-treated fibres retain β-catenin staining in specific foci of the synaptic region. Collectively, our data reveal that lithium treatment differentially affects the stability of postsynaptic receptors in normal and denervated neuromuscular synapses in vivo, thus providing novel insights into the regulatory effects of lithium on synaptic organization and extending its potential therapeutic use in conditions affecting the peripheral nervous system.

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Section: Resultsmentioning

confidence: 97%

Lithium causes differential effects on postsynaptic stability in normal and denervated neuromuscular synapses

Zelada

Barrantes

Henríquez

2021

Sci Rep

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“…As an alternative strategy to approach this idea in culture conditions closer to a physiological situation, we setup nerve-muscle co-cultures. With this aim, C2C12 myoblasts were seeded onto polyornithine/laminin-coated dishes to induce the formation of complex postsynaptic structures, similar to those observed in vivo [ 18 , 32 ]. Once differentiated into myotubes, NSC-34 cells were seeded on top of differentiated myotubes for 24 h and subsequently treated with neural agrin ( Figure 4 A).…”

Section: Resultsmentioning

confidence: 99%

“…Our previous findings showed that Wnt3 obtained from conditioned media of transfected heterologous cells has the ability to collaborate with agrin to induce AChRs clustering in C2C12 myotubes [ 7 ]. In our current studies, we conducted experiments to better mimic a physiological condition by: (i) using cultures of C2C12 myotubes on laminin-coated dishes that resemble the NMJ extracellular environment and induce the formation of complex postsynaptic structures [ 18 , 32 ], and (ii) establishing co-cultures of these myotubes with NSC-34 cells overexpressing Wnt3. We found that when co-cultured with Wnt3-expressing NSC-34 cells in the presence of agrin, C2C12 myotubes exhibited not only an evident increase in acetylcholine receptors microclusters but also formed bigger complex postsynaptic structures.…”

Section: Discussionmentioning

confidence: 99%

“…Remarkably, Wnt3-expressing NSC-34 cells also increased the abundance of complex postsynaptic structures assembled onto laminin-coated surfaces, either plaques or pretzel-like aggregates. These results show that, along with enhancing the acetylcholine receptor clustering ability of the neural-derived protein agrin [ 7 ], Wnt3 could also potentiate the aggregating effect of laminin, a key extracellular matrix regulator of the formation of mature-like postsynaptic apparatuses [ 18 , 32 ]. These results further highlight the potential physiological role of motor neuron-derived Wnt3 on postsynaptic assembly and maintenance at the vertebrate NMJ.…”

Section: Discussionmentioning

confidence: 99%

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

et al. 2021

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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.

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Response characteristics and optimization of electroporation: simulation based on finite element method

Zhou

Yan

Liu

2021

Electromagnetic Biology and Medicine

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Efficient gene transfer into primary muscle cells to analyze nerve-independent postsynaptic organization in vitro

Cited by 3 publications

References 26 publications

Lithium causes differential effects on postsynaptic stability in normal and denervated neuromuscular synapses

Lithium causes differential effects on postsynaptic stability in normal and denervated neuromuscular synapses

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

Response characteristics and optimization of electroporation: simulation based on finite element method

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