AbstractCu/Zn superoxide dismutase (SOD1) is a cytoplasmic antioxidant enzyme, which, when mutant in humans, is linked to familial cases of the motor neurodegenerative disease amyotrophic lateral sclerosis (ALS). The Drosophila SOD1 gene (Sod) shares a highly conserved sequence with the human homolog, and this study includes examinations of the established hypomorphic n108 allele (Sodn108), alongside a knock-in construct of the G85R allele found in human ALS patients (SodG85R). In addition to previously documented decreased adult lifespan and attenuated motor function, we show that Sod mutant Drosophila can display significant mortality during larval and pupal development. Immunostaining of neuronal membrane at neuromuscular synapses in Sod mutant larvae revealed presynaptic terminals of abnormal morphology, with incompletely segregated and enlarged synaptic boutons along the motor terminal branches, in which vital staining indicated mitochondrial aggregation. We demonstrate strong genetic interactions between SodG85R and the axon transport-linked Pk mutants PkPk and PkSple in larval NMJ morphology and neuromuscular transmission. Intracellular recordings of larval excitatory junction potentials (EJPs) demonstrate enhanced EJP size in the double-mutant of PkPk and SodG85R. To examine synaptic terminal excitability, maintained by Ca2+ channel action and independent of Na+ channel function, we used the NaV blocker TTX, along with the KV1 blocker 4-aminopyridine (4-AP) and the commonly used broad-spectrum K+ channel blocker tetraethylammonium (TEA). We were able to induce prolonged “plateau-like” EJPs, which were further extended in Pk mutants and Pk;Sod double-mutants. These observations were corroborated with focal EJP recording from individual boutons. Altogether, this study highlights alterations in synaptic morphology and function at a developmental stage prior to neurodegeneration and death of Sod mutant organisms, along with a potential role of axonal transport in the maintenance of neuronal health.