Levels of Acid Sphingomyelinase (ASM) in Caenorhabditis elegans in Microgravity

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease characterized by a loss of motor neurons and, as a result, skeletal muscle atrophy (van Es et al., 2017). Muscle atrophy is the decrease in muscle size/mass that results from loss of contractile and structural proteins and muscle cells (Brooks and Myburgh, 2014; Fanzani et al., 2012). The progressive motor neuron loss, denervation-induced and primary muscle dysfunction and wasting in ALS culminates in paralysis and death (van Es et al., 2017; Loeffler et al., 2016). Unfortunately, there are currently no effective curative treatments or therapeutic strategies for ALS (van Es et al., 2017).

Muscle atrophy is prevalent across many chronic diseases (Powers et al., 2016a), in individuals subjected to prolonged bedrest and immobility (Brooks and Myburgh, 2014), and in astronauts in spaceflight (Fitts et al., 2010). In fact, the neuromuscular system is seen as one of the most heavily impacted physiological systems in microgravity (Fitts et al., 2001). Muscle atrophy in microgravity has been previously linked to a lack of exercise opportunities (Fitts et al., 2010). However, the use of resistive exercise countermeasures programs by astronauts was only able to partially mitigate muscle atrophy in microgravity (Fitts et al., 2010; Tanaka et al., 2017). Given the engineering challenge to generate artificial gravity in spaceflight, and the inability of resistance exercise to prevent atrophy in microgravity, it is essential to continue to delineate the molecular mechanisms underpinning muscle loss in space to enable the development of effective interventions to preserve and/or restore muscle mass. We were provided an opportunity by the Student Spaceflight Experiments Program (SSEP), a space education program aimed at students from elementary school to college level administered through the National Center for Earth and Space Science Education (NCESSE) and the Arthur C. Clarke Institute for Space Education, to conduct an experiment on the International Space Station (ISS). We decided to investigate the underlying causes of muscle atrophy in microgravity. This research may bring us one step closer to developing an effective treatment for muscle wasting and weakness in both astronauts during spaceflight and in individuals with chronic diseases such as ALS.

Oxidative stress contributes to the development of skeletal muscle atrophy in both chronic disease states and inactivity via a myriad of cellular signaling networks (Cutler et al., 2002; Powers et al., 2016b), Microgravity has been shown to induce oxidative stress (Takahashi et al., 2017). Sphingomyelin and ceramide accumulation have been linked to both ALS pathogenesis and to oxidative stress (Cutler et al., 2002). Furthermore, in the SOD^G86R ALS mouse model, there is evidence of increased gene expression of some sphingomyelinasesenzymes that catalyzes the hydrolysis of sphingomyelin to phosphorylcholine and ceramide (Henriques et al., 2018). This could contribute further to ceramide accumulation. Acid sphingomyelinase (ASM) levels are elevated and activity is increased in the presence of oxidative stress, infection, and inflammation (Kornhuber et al., 2015). However, ASM also releases ceramide, which in itself induces oxidative stress (Jana et al., 2009). These data together suggest that ASM dysregulation may be involved in the pathology of ALS and muscle wasting of spaceflight

We therefore undertook a study to determine ASM levels in Caenorhabditis elegans (C. elegans), a small, yet complex organism with a neuromuscular system: (Szewczyk and Jacobson, 2005). C. elegans is a small nematode that is very similar genetically to humans (Altun and Hall, 2006). In fact, almost 40% of its genes are closely related to those of humans and C. elegans is recognized as a suitable model for studying human disease in space (Adenle et al., 2009). C. elegans has three ASMs, but this study focuses on ASM-1 and ASM-2. Both ASM-1 and ASM-2 are 30% homologous to the human ASM (Lin et al., 1998).

In this study, C. elegans were sent to the ISS to experience microgravity, while control C. elegans remained on Earth. We hypothesized that the worms on the ISS would develop muscle atrophy and experience increased ASM levels compared to the ground experiment. Atrophy extent was determined by morphometric analyses. ASM expression was determined by measuring protein levels. Overall, this study aimed to elucidate the role of ASM in muscle atrophy in microgravity, which may have additional implications for diseases characterized by muscle disuse and wasting.

This is, to our knowledge, the first study completed to assess ASM protein expression in C. elegans in microgravity. The purpose of the experiment was to gain a better understanding of the molecular regulation of muscle atrophy in microgravity, which may also provide insight into the mechanisms of atrophy in disease. We hypothesized that oxidative stress and altered ASM expression are involved in the induction of muscle atrophy that results from both sustained exposure to microgravity, as is seen in astronauts, and from ALS. We predicted that ASM levels would be elevated in C. elegans exposed to microgravity, as sphingomyelinase gene expression levels and ceramides have been found to increase in mouse models of ALS (Henriques et al., 2018; Cutler et al., 2002) We also aimed to test whether the nematodes’ muscle mass would be impacted by the microgravity conditions.

We found that C. elegans exposed to microgravity were longer, had greater cross-sectional areas, and had markedly reduced ASM-1 and ASM-2 expression compared to the control worms. These are striking and unanticipated results, contrary to our hypothesis and previous literature. Muscle atrophy is observed in humans in spaceflight and previous studies have shown similarly that C. elegans in microgravity experience decreased rates of myogenic transcription and have lower muscle density (Higashibata et al., 2006). Higashibata (Higashibata et al., 2016) also reported a decrease in length and fat accumulation in the space flown C. elegans. Furthermore, ASM-2 gene expression analyses using cDNA microarrays from International C. elegans first experiment (ICE-FIRST) (Selch et al., 2008) and C. elegans RNai space experiment (CERISE) (Higashitani et al., 2009) showed a 1.3 fold increase and a 1.1 fold decrease, respectively, although these results were not statistically significant (unpublished data, personal communication with Nathaniel J. Szewczyk).

Interestingly, Kim and Sun (2012) report that inactivation of the ASM homologs leads to slower reproduction and increased longevity of C. elegans. Given the lower expression levels of both ASM-1 and ASM-2 in the space worms, we speculate this may have increased their longevity, resulting in an increase in size compared to the control worms. Furthermore, slower reproduction meant that there were fewer worms in the space tube than in the control tube. As a result, each space worm had greater access to nutrients and oxygen, which might also explain why the space worms were able to grow larger. The reason for the decrease in ASM levels in space worms relative to the control worms is not known. ASM expression and activity is influenced by stress (Chung et al., 2016), and stressors may have impacted the two cohorts of worms differentially, but this was not assessed in our study.

Of interest, we were able to demonstrate that a population of C. elegans can survive for 10 weeks within the confines of a 17 cm FME; to our knowledge this is a novel finding. A previous study completed as part of the SSEP program did not yield live C. elegans upon return to Earth, due to an error in feeding the worms during spaceflight. This experiment concluded that the FME could not produce viable populations for post-flight analysis on extended missions such as the SSEP missions (Warren et al., 2013). With the return of 72,050 live C. elegans from the ISS, our study demonstrates that nematode populations can survive with limited oxygen and nutrients for at least 70 days. Not only did the worms survive space travel, but the population grew to more than fourteen times its original size. Therefore, C. elegans may be used as a model organism for future prolonged microgravity experiments in FME tubes.

There were a number of limitations to our study, as a result of regulations set by NanoRacks, NASA, and SpaceX. Only one tube could be sent to space, thereby permitting no experimental replicates, which limited the power of our experiment. Although we could not send multiple FME tubes to the ISS, we maximized the number of C. elegans within the tube to account for variability between worms. Additionally, the experiment needed to be performed in an FME, which was difficult to use. It was prone to leakage, contamination, and overall wear and tear. Although preliminary testing conducted by us to optimize experimental conditions allowed us to determine the most effective way to seal, subtle leakage or contamination during spaceflight could have impacted our results. In order to prevent leakage, two twist ties, gel, and a plastic cap were placed on each end of the tube; however, 1.5 mL of liquid was still lost due to leakage during the trip.

There were also several limitations of the experimental design. We did not test for the third ASM isoform, ASM-3, which may also affect the lifespan of the nematodes and their oxidative stress levels. We did not test for ASM-3 because a commercially produced antibody was not available. The anti-ASM antibody used was generated against the human ASM, and while it recognized proteins at the correct molecular weight of ASM-1 and ASM-2, it also recognized several other bands of unknown significance. These bands may have been due to non-specific reactivity or reactivity with degradation products. It is unclear why these bands were only seen in the ground control worms, given that GAPDH levels indicated equal loading of ground and space worm lysates. Furthermore, we did not evaluate oxidative stress, ASM-1 or 2 activity, or evaluate behavioral changes in the worms, which may be influenced by oxidative stress and could have served as a surrogate marker (Possik and Pause, 2015). Although this data may have enriched our knowledge, we were mainly focused on the impacts of microgravity on ASM expression. Lastly, C. elegans size is in part dependent on maturation stage. Notably we did not evaluate maturation stage of the space or ground worms, which may have confounded our morphometric measurements.

In summary, as high school students working within the SSEP program, we have importantly demonstrated that C. elegans can survive for at least 70 days with limited nutrients, space, and oxygen and are therefore a feasible and effective model for spaceflight and for SSEP experiments. We also demonstrated a statistically significant difference in expression of ASM between ground and space worms, although the underlying mechanisms are not known. Future research should aim to further evaluate the role of ASM in muscle atrophy in both spaceflight and in diseases such as ALS, as this may enable the identification of targets and strategies for therapeutic manipulation.