Improving human health in space

New therapeutic approaches are required to battle deteriorating health and mission performance during spaceflight. Using worms as model organisms, RNA interference which is used as a method to silence gene expression, emerges as a promising new therapeutic tool against spaceflight induced pathophysiologies.

Human exploration of space is one of the main aims of the world’s space agencies. However, the success of long-term missions into outer space will depend upon overcoming the negative effects of spaceflight on crew health. Indeed, it is known from past missions to the International Space Station (ISS) that exposures to microgravity and/or space radiation can have deleterious effects on astronauts. After only seven days of spaceflight, astronauts start to suffer from conditions such as impaired immune function, loss of bone density and loss of skeletal muscle; the latter of which has been predicted to reach up to 40% during long-term missions. This decline in health can lead to impaired mission performance, increased risk of injury and morbidity and a prolonged recovery from space flight.

Several years ago, space agencies such as NASA revised their standards for astronaut health and introduced countermeasures such as exercise and improved nutrition in space. These countermeasures were based on observations on Earth where exercise and nutrition were shown to combat some muscle protein loss caused by muscle disuse due to bed rest or immobilisation; both are thought to be comparable to muscle loss due to microgravity. However, it is not known if these strategies will be sufficient to prevent muscle loss and other space-induced pathophysiologies as initial trials have produced varied results. The discovery and validation of other prevention therapies is therefore necessary especially for future long-term missions.

Scientists from the University of Nottingham, UK and the Tohoku University in Sendai, Japan have recently published results from the space experiment CERISE (C. elegans RNA interference Space Experiment) in association with the Japanese and American Space agencies1. They described the use of the nematode Caenorhabditis elegans (C. elegans) as model organisms to test potential therapeutics against spaceflight-adapted physiologies. The aim of the research was to determine if RNA interference (RNAi), a widely used technique to silence expression of a specific gene, also works in space and if it can potentially prevent muscle loss.

RNAi is an evolutionarily conserved mechanism which controls gene expression and acts as an innate immune defence mechanism against pathogenic nucleic acids such as viruses and other transposable elements. It was first discovered in C. elegans that RNAi is mediated by the presence of exogenous or synthetically produced double stranded RNA (dsRNA)2. In order to silence a targeted gene, dsRNA is cleaved into ~20 nucleotide long, small-interfering RNA (siRNA) by an RNA splicing protein called Dicer. The siRNA then attaches to complementary mRNA at the RNA induced silencing complex (RISC) leading to degradation of the mRNA. Similarly to siRNA, microRNA (miRNA) which are ~21 nucleotide long non-coding RNA, serve as posttranscriptional silencers and form the endogenous RNAi pathway.

RNAi is therefore utilised as a tool to silence gene expression in order to study gene function and related regulatory mechanisms. Furthermore, RNAi is studied as a potential gene silencing tool in the fight against diseases such as cancer in which overexpression of specific genes results in uncoordinated cell growth. Several RNAi-based clinical trials are conducted on Earth but much more work will be necessary to develop RNAi into a therapy3.

Space imposes different environmental conditions to Earth, such as radiation and microgravity. Indeed, it was observed previously that lymphocytes and T-cells which are components of the immune system were down-regulated in astronauts. The scientists were therefore concerned that the space environment could affect RNAi, because of its role as an immune defence mechanism. Furthermore, there was the possibility that radiation could affect the stability of dsRNA as it is very unstable naturally, potentially influencing RNAi efficacy. Thus, the use of RNAi as a potential tool against spaceflight-induced pathophysiologies needed to be evaluated.

The 1mm long nematode C. elegans has been serving as a model organism in space research for more than 10 years with specific focus on the effects of cosmic radiation and spaceflight upon muscle. On Earth, C. elegans has already been established as a genetic model to gain insight into muscle development, myofibril assembly, muscular dystrophies and protein degradation due to its high similarity to human muscle structure and many evolutionarily well preserved biological processes. C. elegans are easy and cheap to cultivate on a bacterial food source and tissue structures and specific proteins can be easily examined in vivo using light and fluorescence microscopy thanks to its transparency.

The most commonly used RNAi technique in C. elegans is the feeding method. E. coli bacteria clones which produce dsRNA of a gene of interest from an integrated plasmid are fed to the worms. Understanding of how the dsRNA reaches the various cells is currently unclear, but reduced gene expression levels and observations of developmental phenotypes such as sterility, body morphology or movement defects confirm RNAi activity in cells.

In order to evaluate the RNAi technique, the researchers sent thousands of worms to the ISS onboard the space shuttle Atlantis on 16th November 2009. C. elegans have a lifecycle of four days but have the ability to arrest during early larvae development due to the absence of food; in this state, larvae can survive up to six months. When food becomes available arrested larvae develop normally into adults within two to three days. Using a culturing bag system, developmentally arrested worms were kept separated from bacterial feeds by clamping the middle of the bag with a U-pin until reaching the ISS. This strategy ensured that acute RNAi experiments could be performed in adult worms and that RNAi experiments could be scheduled.

On the ISS, the experiments were conducted in the Cell Biology Experiment Facility of the Japanese Experiment Module Kibo. Upon reaching microgravity in the module, the experiment was started by an astronaut pulling the U-pin from the bag and thus feeding worms with either bacteria or RNAi bacteria clones for four or eight days. Worms were then frozen and stored at -80°C until post-flight analysis back on Earth. Simultaneously, the same experiments were performed for ground controls on Earth.

At post-flight analysis, the scientists evaluated the conservation of the RNAi machinery by comparing gene expression levels of major RNAi machinery components and miRNAs to ground controls. Results demonstrated that the majority of expression levels were unaltered indicating that the RNAi machinery was still intact and not affected by changes in immunity responses and metabolism due to space flight.

To investigate if the RNAi mechanism was functioning, the researchers utilised a previously evaluated worm strain containing a green fluorescent protein (GFP) reporter which labels a protein localising to chromosomes in oocytes and embryos. Upon RNAi treatment against GFP or the chromosome-localising protein in space, reduction in GFP expression levels and observations of abnormal chromosomal GFP localisation were in accordance with results on Earth. This indicated that the RNAi mechanism seems to be preserved in gonad tissue. In order to show that RNAi is effective in other tissues the researchers investigated the effects of space on muscle tissue.

Muscle is a very adaptable tissue and begins to waste when it is not used such as in a microgravity environment during spaceflight. Muscle contains about 50% of the body’s protein making it a main metabolic regulator; therefore major muscle loss can negatively affect astronauts’ metabolism and health. To test if RNAi could prevent the loss of muscle mass due to spaceflight, the researchers treated worms with dsRNA against two lysosomal cathepsins, enzymes known to degrade muscle proteins.

RNAi treatment resulted in increased levels of the muscle protein a-actin compared to non-treated animals in space and on Earth. This result indicates that RNAi against cathepsins can reduce lysosomal protein degradation activity during spaceflight and on Earth. However, this analysis was based upon lysosomal degradation induced during biochemical sample preparation (the researchers used a protease inhibitor cocktail with known activity against all known proteolytic systems apart from the lysosomal system). Thus while the researchers showed that RNAi can work to decrease protein degradation it has not yet been demonstrated that muscle protein degradation increases during spaceflight.

The results demonstrate that RNAi works effectively in various tissues in space. Therefore, this research may serve as a foundation to investigate the use of RNAi as a possible therapeutic method against space-adapted physiologies in several tissues.

Space on the ISS is restricted and research is very expensive therefore experiments in space need to be efficient and they cannot usually be repeated. Experimental equipment for C. elegans is compact as worms can be cultivated in liquid. Experiments can be automated and do not need constant supervision which is advantageous for the experiments success as astronauts are not experienced scientists. C. elegans also allow the observation of cell and tissue specific defects in vivo using microscopes while still in space which is extremely difficult to do in mammalian models such as mice or rats.

This study supports the application of C. elegans as a model organism to study space effects on organism health and fundamental biological processes and to discover new therapies, before transferring these findings to human. If in the future, the space agencies move on further into outer space, worms could be used as a first organism from Earth to measure if long-term missions and outer space environment will have life threatening effects on human.

Author: Susann Lehmann PhD student in the lab of Dr Nathaniel J Szewczyk at the University of Nottingham

References:

1. T. Etheridge et al. The Effectiveness of RNAi in Caenorhabditis elegans Is Maintained during Spaceflight, 6 PLOS ONE.e20459, e20459 (2011)
2. A. Fire et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans, 391 NATURE.806, 806-811  (1998)
3. B. L. Davidson & P. B. McCray, Jr. Current prospects for RNA interference-based therapies, 12 NAT REV.GENET.329, 329-340  (2011)