NEMUCO: The In Vitro 4D NMJ as an Innovative Powerful Tool to Study Nerve and Muscle Cell–Cell Communication

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.