Modelling FUS Mislocalisation in an In Vitro Model of Innervated Human Muscle

Degeneration of distal axons and neuromuscular junctions is an early feature in the pathology of amyotrophic lateral sclerosis (ALS), which culminates in motor neuron loss due to axon retraction and muscle atrophy. The complex interactions in the pathogenesis of ALS between motor neurons, muscle cells and accompanying glia require an appropriate experimental model. Here, we have defined a co-culture model based on human myotubes innervated by neurons from embryonic rat spinal cord explants to investigate the pathology and treatment of ALS. This model was first characterised for endogenous expression and distribution of ALS-related proteins TDP-43 and FUS. Then, wild-type FUS and its mutants were introduced into these co-cultures to determine how FUS defects in nuclear transport modulate the pathological conditions. FUS-bearing plasmids were introduced by classical transfection and electroporation, as novel approaches to deliver plasmids into explants, and their cellular distributions were characterised. Endogenous nuclear expression of TDP-43 and FUS was observed in explants and myoblasts/myotubes. After transfection, wild-type FUS was expressed in nuclei of myoblasts, myotubes and explants, although with low transfection rates. Following successful electrotransfection into explants, the localisation of wild-type FUS was nuclear, and it was detected in neurons, astrocytes, Schwann cells and oligodendrocyte precursors, whereas the FUS∆Y, FUSY526A and FUSY526E mutants were cytoplasmic, and the FUSY526F mutant was nuclear and cytoplasmic. This co-culture model is applicable to the study of neuronal and non-neuronal cell contributions to ALS and other neurodegenerative diseases, and it can be used to investigate drug targets amenable to intervention.