Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the loss of motoneurons (MNs), and despite progress, there is no effective treatment. A large body of evidence shows that astrocytes expressing ALS-linked mutant proteins cause non-cell autonomous toxicity of MNs. Although MNs innervate muscle fibers and ALS is characterized by the early disruption of the neuromuscular junction (NMJ) and axon degeneration, there are controversies about whether muscle contributes to non-cell-autonomous toxicity to MNs. In this study, we generated primary skeletal myotubes from myoblasts derived from ALS mice expressing human mutant SOD1G93A (termed hereafter mutSOD1). Characterization revealed that mutSOD1 skeletal myotubes display intrinsic phenotypic and functional differences compared to control myotubes generated from non-transgenic (NTg) littermates. Next, we analyzed whether ALS myotubes exert non-cell-autonomous toxicity to MNs. We report that conditioned media from mutSOD1 myotubes (mutSOD1-MCM), but not from control myotubes (NTg-MCM), induced robust death of primary MNs in mixed spinal cord cultures and compartmentalized microfluidic chambers. Our study further revealed that applying mutSOD1-MCM to the MN axonal side in microfluidic devices rapidly reduces mitochondrial axonal transport while increasing Ca2 + transients and reactive oxygen species (i.e., H2O2). These results indicate that soluble factor(s) released by mutSOD1 myotubes cause MN axonopathy that leads to lethal pathogenic changes. C1 [Martinez, Pablo; Abarzua, Sebastian; Bustos, Fernando J.; van Zundert, Brigitte] Univ Andres Bello, Inst Biomed Sci ICB, Fac Med, Santiago, Chile. [Martinez, Pablo; Abarzua, Sebastian; Bustos, Fernando J.; van Zundert, Brigitte] Univ Andres Bello, Fac Life Sci, Santiago, Chile. [Silva, Monica; Jaimovich, Enrique] Univ Chile, Fac Med, Ctr Exercise Metab & Canc, Inst Ciencias Biomed, Santiago, Chile. [Tevy, Maria Florencia] Univ Atacama, Fac Med, Copiapo, Chile. [Constantine-Paton, Martha] MIT, McGovern Inst Brain Res, Dept Brain & Cognit Sci, Cambridge, MA USA. [Bustos, Fernando J.; van Zundert, Brigitte] Millennium Nucleus Neuroepigenet & Plast EpiNeuro, Santiago, Chile. [van Zundert, Brigitte] Univ Massachusetts Chan Med Sch UMMS, Dept Neurol, Worcester, MA 01655 USA. C3 Universidad Andres Bello; Universidad Andres Bello; Universidad de Chile; Universidad de Atacama; Massachusetts Institute of Technology (MIT); University of Massachusetts System; UMass Chan Medical School