Homocysteine sensitizes the mouse neuromuscular junction to oxidative stress via nitric oxide

Homocysteine, a redox-active metabolite of the methionine cycle, is of particular clinical interest due to its association with various neurodegenerative diseases including Amyotrophic Lateral Sclerosis. It has been previously established that homocysteine exacerbates damage to motor neurons from reactive oxygen species (ROS) such as hydrogen peroxide. To assess the role of homocysteine at the mammalian neuromuscular junction, neurotransmission was monitored via electrophysiology at the mouse Epitrochleoanconeus (ETA) muscle. Preparations were pre-incubated in homocysteine prior to inducing ROS and recordings were taken before and after ROS treatment. In this study, homocysteine was observed to sensitize the neuromuscular junction to ROS-induced depression of spontaneous transmission frequency, an effect we found to be mediated via an NMDA receptor and nitric oxide. The NMDA receptor antagonist DL-2- Amino-5-phosphonopentanoic acid prevented the depression from homocysteine-induced sensitization to oxidative stress. Disrupting nitric oxide activity with either the NOS I antagonist Nω-Nitro-L-Arginine methyl ester hydrochloride or the NO scavenger 2-(4-Carboxyphenyl)-4,4,5,5-tetramethyl- imidazoline-1-oxyl-3-oxide potassium salt also prevented the depression. Moreover, replacing homocysteine with the exogenous NO donor Diethylamine NONOate diethylammonium was sufficient to reconstitute the effects of homocysteine-induced sensitization to ROS. Interestingly, a novel secondary effect was observed where homocysteine itself depresses quantal content, an effect found to be mediated by NMDARs independently of nitric oxide and ROS. Collectively, these data present a novel model of two distinct pathways through which homocysteine alters neurotransmission at the neuromuscular junction. Characterizing homocysteine’s mechanism of action is of particular clinical relevance as many treatments for ALS are centered on mitigating homocysteine-induced pathologies.