Description

Free radicals, such as the superoxide anion (O₂^–) impart oxidative stress upon an animal and are thought to be a major contributing factor to age-related changes in the nervous system (Finkel & Holbrook, 2000; Harman, 1956; Harman, 1981). In eukaryotes, the cytosolic enzyme Cu²⁺/Zn²⁺ Superoxide Dismutase (in Drosophila encoded by Sod1, originally cSOD, Campbell et al., 1986) is an important free radical scavenger that converts superoxide into hydrogen peroxide. Drosophila Sod1 loss-of-function mutants exhibit elevated levels of reactive oxygen species (ROS) combined with a drastically shortened adult lifespan (median ~11 d. vs 50 d. for wild-type flies at 25 °C, Phillips et al., 1989) and reduced locomotor ability in larvae (Şahin et al., 2017) and adults (Ruan & Wu, 2008). Coupled with these longevity and behavioral phenotypes, several neuromuscular deficits have recently been identified in Sod1 loss-of-function mutants including deranged nerve and synapse morphology in larvae (Milton et al., 2011) and adults (Agudelo et al., 2020; Şahin et al., 2017), as well as disrupted neurotransmission along the giant-fiber (GF) jump-and-flight escape circuit (Iyengar et al., 2020; Iyengar, 2016; Ruan, 2008). Given the striking phenotypes, a question that arises is: To what extent the straightforward assumption holds that aspects of the Sod1 phenotypes can be recapitulated by pharmacologically induced oxidative stress?

Here, we compare neuromuscular physiology in Sod1 loss-of-function (‘null’) mutants to wild-type (WT) flies with elevated oxidative stress levels induced by paraquat (PQ) feeding. A widely used herbicide, PQ is a well-studied drug often utilized experimentally to generate superoxide anions and induce oxidative stress in Drosophila (Parkes et al., 1993; Shukla et al., 2014). We examined synaptic transmission at the abdominal neuromuscular junction (NMJ) of 3^rd instar Sod1 larvae to WT larvae fed on PQ-containing medium from egg hatching. In adults, we compared the performance of the GF escape pathway (Engel & Wu, 1992) in Sod1 mutants to PQ-fed WT flies. The PQ feeding concentrations chosen for this study (5 or 10 mM throughout the larval stage; and 10 mM in adults for 4 d) were reported to induce lethality and behavioral abnormalities in WT flies within a 3 – 7 d window (Arking et al., 1991; Jahromi et al., 2013). Over this period, we indeed observed a marked loss of motor coordination. Flies often “stumbled” while walking and occasionally exhibited wing-buzzes upon gentle “tapping” of the vial. The PQ concentrations selected for exposing larvae throughout their development was near lethality levels, beyond which few larvae survived. Nevertheless, our neurophysiological findings reveal that PQ-fed WT individuals clearly do not replicate all Sod1 phenotypes in both larval and adult physiological recordings.

At the larval NMJ, we found Sod1 mutant larvae displayed a striking phenotype which was absent in WT counterparts (Figure 1A). Although an initial nerve stimulation produced similar excitatory junctional potentials (EJPs, analogous to EPSPs) in WT and Sod1 larvae (lower traces), after repetitive stimulation (at ~15 Hz, upper traces), we observed extra ‘humps’ in the Sod1 waveforms indicative of supernumerary EJPs (representative EJPs from a homozygous Sod1ⁿ¹⁰⁸ and hetero-allelic Sod1ⁿ¹⁰⁸/Sod1^X39 individuals are shown). These abnormal events indicate the stimuli recruit secondary action potentials in the motor nerve (Ueda & Wu, 2009) and suggest a form of nerve hyperexcitability in Sod1 mutants. In contrast, PQ-fed WT flies displayed EJPs that were remarkably similar to control diet-fed counterparts in terms of shape and amplitude. Indeed, across the population of larvae, extra ‘humps’ in the EJP waveform were commonly observed in Sod1 larvae but were exceedingly rare in PQ-fed WT or control-fed WT flies (Figure 1B).

To compare the effects of PQ feeding and Sod1 mutations on adult nervous system function, we examined stimulus-response relationships of the GF pathway. In this circuit, established visual and mechanosensory inputs trigger spiking in the GF neuron in response to visual looming or air pressure stimuli (Card & Dickinson, 2008; Lehnert et al., 2013; Mu et al., 2014; Trimarchi & Schneiderman, 1995). The descending GF axons project directly to the tergotrochanteral (TTM, ‘jump muscle’) motor unit and indirectly via the peripherally synapsing interneuron (PSI) to the dorsal longitudinal muscle (DLM, ‘flight muscle’) motor unit (Gorczyca & Hall, 1984; King & Wyman, 1980). Direct electrical stimulation of the GF neuron triggers an action potential in the DLM with a stereotypic latency in WT flies (~1.4 ms, Figure 1C), known as ‘short-latency’ responses (Engel & Wu, 1996). Consistent with previous observations (Iyengar et al., 2020), we found Sod1 flies displayed an overall increase in latency with a wider variability compared to WT counterparts (Figure 1C-D). However, PQ-fed WT flies did not show such retarded responses; instead latencies displayed were remarkably similar to control WT individuals. We also examined the ability of the GF pathway to follow trains of high-frequency stimulation (20 – 200 Hz) in PQ-fed flies and Sod1 mutants (Figure 1E). At stimulation frequencies higher than 100 Hz, we found Sod1 displayed a progressively worse ability to follow stimulation compared to WT counterparts (largely consistent with observations using a twin-pulse refractory protocol, c.f. Iyengar et al., 2020). However, across all frequencies, PQ-fed WT flies displayed a similar response rate compared to control diet-fed individuals.

Our observations at the larval NMJ and along the GF pathway in adults reveal clear and consistent defects in neurotransmission among Sod1 loss-of-function mutants. It should be noted that mutant flies harboring an amyotrophic lateral sclerosis (ALS)-associated allele (G85R) has been reported to display a collection of neuromuscular defects, including NMJ overgrowth and moderately increased spontaneous miniature EJPs, which can be further compared with PQ-feeding effects in future studies (Held et al., 2019). The Sod1 alleles we studied, i.e. Sod1ⁿ¹⁰⁸, Sod1^X39 and Sod1²¹, were independently isolated (Phillips et al., 1989; Şahin et al., 2017). Thus, it is most likely Sod1 disruption, rather than contributions from secondary ‘background’ mutations, gives rise to the phenotypes. We observed poor motor coordination and lethality, indicating our PQ-feeding indeed produced effects consistent with previous reports (Arking et al., 1991). Notably, even though the larval NMJ and adult GF pathway are remarkably robust systems, studies from our group and others have demonstrated clear electrophysiological alterations in Sod1 loss-of-function (this study; see also: Watson, et al., 2008, Iyengar, et al., 2020). In contrast, pharmacological manipulation of oxidative stress levels with standard PQ-feeding protocol failed to recapitulate Sod1 mutant phenotypes, suggesting the neuromuscular deficits in Sod1 flies described here do not arise by a simple overall elevation of superoxide levels. One possible contributing factor is altered regulation of superoxide anions in specific cell types or subcellular compartments leading to the complexity or diversity of Sod1 mutant phenotypes. Alternatively, mutations in Sod1 or PQ-exposure can induce different patterns of downstream adjustments involving altered activity of other redox enzymes (Missirlis et al., 2003) and of different 2^nd messenger signaling pathways such as BMP signaling (Berke et al., 2013; Held et al., 2019), Nrf2/KEAP1 and/or NF-KB, (Sies et al., 2017; Wang et al., 2018).