Peripheral nervous system neurons

Sensory neurons

Sensory trigeminal ganglion neurons grow well on laminin 411, but not on laminin 211 (Fried, 2005). Adult dorsal root ganglion (DRG) neurons are differentially regulated by laminins where laminin isoforms 111 and 511 show the highest stimulation of neurite outgrowth (Plantman, 2008). Laminin 332 suppresses the mechanotransduction and axonal branching of cultured DRG sensory neurons. (Chiang, 2011)

Motor neurons and neuromuscular junctions

Motor neurons in culture extend longer neurites on Biolaminin 211 compared to when cultured on Biolaminin 411 (Wallquist, 2005). Wallquist and colleagues also showed that loss of α4 (laminin isoforms 411 and 421) leads to a disturbance in radial sorting and impaired myelination (Wallquist, 2005; Chand, 2017).

Motor neurons form specialized synapses with skeletal muscle fibers called neuromuscular junctions (NMJs). The function of this type of synapse is to transmit signals from the central nervous system to muscles and thus stimulate their contraction. Muscle cells form a thick ECM around the fiber that contains various laminins, collagens, fibronectin, and other glycoproteins. The basal lamina (BL) at the synaptic cleft has a specific molecular composition that contains laminin α4, α5, and β2 isoforms that are mostly absent in extrasynaptic regions of muscle fibers. These ECM components are crucial for the proper development of NMJs and it has been shown that the laminin-dystroglycan interaction is crucial for regulating NMJ developmental remodeling. Laminin 211 is present together with laminin 221, 421 and 521 at neuromuscular junctions and ensures correct localization of pre- and postsynaptic specializations (Sanes and Lichtman, 2001; Patton, 2001; Domogatskaya, 2012).

Little is known about the mechanisms of postsynaptic machinery remodeling in vivo. Mouse laminin-111 has been routinely used to induce AChR clustering and is the only in vitro system where the AChR clusters undergo developmental remodeling that resembles mature NMJs, providing the model to study the underlying mechanisms. In a publication in Scientific Reports (Pęziński, 2020) the authors presented an improved protocol for culturing C2C12 muscle cells that reproducibly promote the formation of complex AChR clusters. The authors tested several laminin isoforms and found that laminin-121, laminin-211, laminin-221, laminin-511, and laminin-521 induced significantly more AChR clusters in C2C12 myotubes than the commonly used laminin-111. Moreover, they found that clusters of postsynaptic machinery that were formed in C2C12 myotubes cultured on laminin-121 and laminin-221 were the most developed. Laminin-421 and laminin-511 were the isoforms that promoted the formation of the most podosome-containing AChR clusters in human primary myotubes. Myotubes that were derived from human primary myoblasts obtained from human biopsies also formed AChR clusters in vitro that underwent the remodeling process, thus demonstrating the potential utility of this methodology for further studies that seek to improve diagnoses of neuromuscular disorders and elucidate their underlying mechanisms. Thus, this novel method may facilitate the identification of novel synaptic regulators, and the high reproducibility of culturing and robust formation of AChR clusters are important prerequisites for establishing high-throughput screening. The protocol is also useful for obtaining and freezing a large number of cell stocks and utilizing cells for experimentation with a constant and low passage number, which significantly increases experimental reproducibility. The method can be implemented in different formats, such as permanox slides, glass surfaces as well as multi-well culturing dishes. Collectively, these results demonstrated an advanced method for culturing myotubes that provide an important basis for high-throughput genetic screening and potential drug development for screening for potential therapeutic targets for neuromuscular disorders.