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, long-term bedrest as either spaceflight analog or actual spaceflight missions, results in a decline of neuromuscular junction (NMJ) structure and function, resulting in muscle mass decline and impaired fine motor control. Similar changes are also observed in several neuromuscular diseases and myopathies with signs and symptoms related to impaired movement control. 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 purpose. Murine NErve (NSC-34 motor neuron) and MUscle (C2C12 myoblast) cell CO-cultures (NEMUCO) were grown on either synthetic or biological three-dimensional (3D) scaffolds. The co-cultures were analyzed using a significant array of molecular and cellular biology tools, based on NMJ-specific molecular marker expression combined with muscle- and nerve-cells 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-positive developing myotubes, representing the first signs of NMJ-like structure assembly in vitro. 3D reconstruction morphometry revealed a 50-nm-wide distance similar to the in vivo native synaptic cleft dimension. It is noteworthy that co-cultured motor neurons showed a trend of increase of SNAP-25 transcription, one of the most important SNARE proteins, whereas co-cultured myotubes showed a slight increase of junctophilin and ryanodine receptor type 1 transcription, both critical proteins of the triadic junction of differentiated muscle fibers. In most respects, neuronal-myotube cell-cell communication contacts in our 3D co-cultures mimicked native 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 during spaceflight conditions.