Description

Reactive oxygen species (ROS) contribute to neuronal degeneration by readily reacting with cellular components, consequently breaking down cellular integrity. Excess ROS often leads to oxidative stress, which results from destabilization of the organism’s ability to control the balance between antioxidants and free radicals (Chandra et al. 2015). The ubiquitin-proteasome system helps to regulate oxidative stress and overall damage to cellular components by forming chains of ubiquitin polypeptides on cellular proteins; these chains then serve as a signal to break down the attached protein (Hershko et al. 1983). Mutation of UBE3B, an E3 ubiquitin ligase, has been found to lead to Blepharophimosis-Ptosis-Intellectual-Disability Syndrome (BPID) in human infants, indicating potential involvement of UBE3B in the regulation of neuronal signaling in the brain (Basel-Vanagaite et al. 2012). The UBE3B protein was also shown to be involved in mitochondrial function and oxidative stress responses in mammalian cells (Braganza et al. 2017). In C. elegans, the oxi-1 gene encodes an ubiquitin ligase homologous to UBE3B (58% amino acid similarity); expression of C. elegans oxi-1 is induced by oxidative stress and is required for proteasomal responses to this stress (Basel-Vanagaite et al. 2012). However, despite the link to neurodevelopmental disorders including BPID, specific roles for UBE3B or oxi-1 in neuronal biology and synaptic function, with or without oxidative stress, have not been explored (Basel-Vanagaite et al. 2012). A second C. elegans gene fshr-1, which encodes a G protein-coupled receptor homologous to a family of mammalian glycopeptide hormone receptors (Cho et al, 2007), is also involved in both oxidative stress responses and neuronal signaling. Specifically, fshr-1 regulates expression of gcs-1, an oxidative-stress response gene (Miller et al. 2015) and was identified in an RNAi interference screen as a gene required for proper structure and function of neuromuscular synapses (Sieburth et al. 2005). Despite data suggesting roles for oxi-1 UBE3B, and fshr-1 in neuronal signaling, which is susceptible to oxidative damage, neither gene has been investigated with regards to neuronal signaling in the presence of oxidative stress. Here, we tested the requirements of both oxi-1 and fshr-1 for their effects on neuromuscular signaling under both normal and oxidative stress conditions in C. elegans. 

Neuronal signaling activity can be measured at the neuromuscular junction in C. elegans, a model synapse, by observing the motility of individual worms in liquid medium in a body bending assay (Nawa and Matsuoka 2012). Under normal conditions, C. elegans mutants lacking expression of either oxi-1 or fshr-1 had reduced motility compared to wild type animals by 11.0% (p < 0.0001= 2.1167E-05) and 11.2% (p < 0.00001= 3.8259E-06), respectively (Figure 1A). Following exposure to oxidative stress conditions (5 mM paraquat) for 48 hours, the mean number of body bends for wild type animals remained constant (normal: 194 body bends/minute, oxidative stress: 199 body bends/minute). However, the motility of oxi-1 mutants compared to wild type N2 worms under oxidative stress conditions in the body bending assay decreased from 173 to 160.5 body bends/minute, a 19.2% reduction compared to the wild type strain (p < 0.001= 1.77E-04) (Figure 1B). An even larger decrease in body bends, from 172.5 to 140.8 body bends/minute, a decrease of 29.2% (p = < 0.00000012.1771E-08) was observed for fshr-1 mutants compared to wild type animals under oxidative stress conditions (Figure 1B). Successful induction of oxidative stress was determined for each body bending assay by assessing whether a separate strain of worms (ldIs3) carrying the oxidative stress responsive reporter gcs-1p::GFP exposed to paraquat alongside oxi-1, N2, and fshr-1 expressed green fluorescence when observed under a fluorescence stereomicroscope.