NEMUCO: The In Vitro 4D NMJ as an Innovative Powerful Tool to Study Nerve and Muscle Cell–Cell Communication
4 affiliations
- 1Institute of Integrative Neuroanatomy, Charité—Universitätsmedizin Berlin, 10115 Berlin, Germany
- 2Section of Padova, CNR-Neuroscience Institute, 35131 Padova, Italy
- 3Department of Life Sciences, University of Trieste, 34127 Trieste, Italy
- 4Department of Biomedical Sciences, University of Padova, 35121 Padova, Italy
Abstract
Chronic muscle unloading, following denervation, aging, disease, or long-term bed rest, whether as a spaceflight analog or during an actual spaceflight mission, results in a decline in structure and function of the neuromuscular junction (NMJ). This decline leads to a decrease in muscle mass and impaired fine motor control. Similar changes are also observed in several neuromuscular diseases and myopathies’ impaired movement control symptoms. Here, we report on a powerful 3D co-culture experimental model developing NMJ-like structures, as a novel in vitro platform for functional/regulatory studies during time (4D co-culture) for basic and translational research purposes. Murine NErve (NSC-34 motoneuron) and MUscle (C2C12 myoblast) cell CO-cultures (NEMUCO) were grown on either synthetic or biological three-dimensional (3D) scaffolds. Morphological analysis obtained through 3D reconstruction in proximity of the cell–cell contact revealed close apposition between NSC-34 motoneurons and C2C12 myotubes. The co-cultures were further analyzed using a significant array of molecular and cellular biology tools, based on NMJ-specific molecular marker expression combined with muscle- and nerve-cell-specific differentiation biomarkers. Specialized cell–cell contacts were present starting at day 2 of co-culture. Triple immunostaining indicated neurofilament-positive axonal nerve terminals approaching clustered α-bungarotoxin-positive nicotinic acetylcholine receptors (nAChRs) in desmin- or fast-type myosin heavy chain (fMyHC)-positive developing myotubes, representing the first signs of NMJ-like structure assembly in our in vitro system. It is noteworthy that co-cultured motoneurons showed a trend of increase in SNAP-25 transcription, one of the most important SNARE proteins, whereas co-cultured myotubes showed a slight increase in junctophilin and ryanodine receptor type 1 (RyR1) transcription, both critical proteins of the triadic junction of differentiated muscle fibers. In most respects, neuronal-myotube cell–cell contacts in our 3D co-cultures mimicked native pre- and postsynaptic NMJ microdomains. Neuronal-myotube co-cultures grown in 3D scaffolds represent a powerful tool for investigating the molecular mechanisms underlying NMJ adaptation and plasticity in muscle myopathies. Moreover, this approach could be adapted for customized miniaturized platforms designed for cellular neurobiology research under spaceflight conditions.
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BibTeX
@article{block2026nemuco,
title = {{NEMUCO: The In Vitro 4D NMJ as an Innovative Powerful Tool to Study Nerve and Muscle Cell–Cell Communication}},
author = {Katharina Block and Gaia Ziraldo and Gabor Trautmann and Sandra Furlan and Abhishek Singh and Sabine Grosser and Martina Gutsmann and Paola Lorenzon and Pompeo Volpe and Imre Vida and Dieter Blottner and Michele Salanova},
journal = {International Journal of Molecular Sciences},
year = {2026},
month = oct,
doi = {10.3390/ijms27208946},
url = {https://www.mdpi.com/1422-0067/27/20/8946},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - NEMUCO: The In Vitro 4D NMJ as an Innovative Powerful Tool to Study Nerve and Muscle Cell–Cell Communication
AU - Katharina Block
AU - Gaia Ziraldo
AU - Gabor Trautmann
AU - Sandra Furlan
AU - Abhishek Singh
AU - Sabine Grosser
AU - Martina Gutsmann
AU - Paola Lorenzon
AU - Pompeo Volpe
AU - Imre Vida
AU - Dieter Blottner
AU - Michele Salanova
PY - 2026
DA - 2026/10/09
JO - International Journal of Molecular Sciences
DO - 10.3390/ijms27208946
UR - https://www.mdpi.com/1422-0067/27/20/8946
AB - Chronic muscle unloading, following denervation, aging, disease, or long-term bed rest, whether as a spaceflight analog or during an actual spaceflight mission, results in a decline in structure and function of the neuromuscular junction (NMJ). This decline leads to a decrease in muscle mass and impaired fine motor control. Similar changes are also observed in several neuromuscular diseases and myopathies’ impaired movement control symptoms. Here, we report on a powerful 3D co-culture experimental model developing NMJ-like structures, as a novel in vitro platform for functional/regulatory studies during time (4D co-culture) for basic and translational research purposes. Murine NErve (NSC-34 motoneuron) and MUscle (C2C12 myoblast) cell CO-cultures (NEMUCO) were grown on either synthetic or biological three-dimensional (3D) scaffolds. Morphological analysis obtained through 3D reconstruction in proximity of the cell–cell contact revealed close apposition between NSC-34 motoneurons and C2C12 myotubes. The co-cultures were further analyzed using a significant array of molecular and cellular biology tools, based on NMJ-specific molecular marker expression combined with muscle- and nerve-cell-specific differentiation biomarkers. Specialized cell–cell contacts were present starting at day 2 of co-culture. Triple immunostaining indicated neurofilament-positive axonal nerve terminals approaching clustered α-bungarotoxin-positive nicotinic acetylcholine receptors (nAChRs) in desmin- or fast-type myosin heavy chain (fMyHC)-positive developing myotubes, representing the first signs of NMJ-like structure assembly in our in vitro system. It is noteworthy that co-cultured motoneurons showed a trend of increase in SNAP-25 transcription, one of the most important SNARE proteins, whereas co-cultured myotubes showed a slight increase in junctophilin and ryanodine receptor type 1 (RyR1) transcription, both critical proteins of the triadic junction of differentiated muscle fibers. In most respects, neuronal-myotube cell–cell contacts in our 3D co-cultures mimicked native pre- and postsynaptic NMJ microdomains. Neuronal-myotube co-cultures grown in 3D scaffolds represent a powerful tool for investigating the molecular mechanisms underlying NMJ adaptation and plasticity in muscle myopathies. Moreover, this approach could be adapted for customized miniaturized platforms designed for cellular neurobiology research under spaceflight conditions.
SN - 1422-0067
ER -