Although muscle atrophy is a serious problem during spaceflight, little is known about the sequence of molecular events leading to atrophy in response to microgravity. We carried out a spaceflight experiment using Caenorhabditis elegans onboard the Japanese Experiment Module of the International Space Station. Worms were synchronously cultured in liquid media with bacterial food for 4 days under microgravity or on a 1-G centrifuge. Worms were visually observed for health and movement and then frozen. Upon return, we analyzed global gene and protein expression using DNA microarrays and mass spectrometry. Body length and fat accumulation were also analyzed. We found that in worms grown from the L1 larval stage to adulthood under microgravity, both gene and protein expression levels for muscular thick filaments, cytoskeletal elements, and mitochondrial metabolic enzymes decreased relative to parallel cultures on the 1-G centrifuge (95% confidence interval (P⩽0.05)). In addition, altered movement and decreased body length and fat accumulation were observed in the microgravity-cultured worms relative to the 1-G cultured worms. These results suggest protein expression changes that may account for the progressive muscular atrophy observed in astronauts.

We have started a space experiment using an experimental organism, the nematode Caenorhabditis elegans, in the Japanese Experiment Module, KIBO, of the International Space Station (ISS). The specimens were boarded by space shuttle Atlantis on mission STS-129 which launched from NASA Kennedy Space Center on November 16, 2009. The purpose of the experiment was severalfold: (i) to verify the efficacy of RNA interference (RNAi) in space, (ii) to monitor transcriptional and post-translational alterations in the entire genome in space, and (iii) to investigate mechanisms regulating and countermeasures for muscle alterations in response to the space environment. In particular, this will be the first study to utilize RNAi in space.

BackgroundOvercoming spaceflight-induced (patho)physiologic adaptations is a major challenge preventing long-term deep space exploration. RNA interference (RNAi) has emerged as a promising therapeutic for combating diseases on Earth; however the efficacy of RNAi in space is currently unknown.Methods Caenorhabditis elegans were prepared in liquid media on Earth using standard techniques and treated acutely with RNAi or a vector control upon arrival in Low Earth Orbit. After culturing during 4 and 8 d spaceflight, experiments were stopped by freezing at −80°C until analysis by mRNA and microRNA array chips, microscopy and Western blot on return to Earth. Ground controls (GC) on Earth were simultaneously grown under identical conditions.ResultsAfter 8 d spaceflight, mRNA expression levels of components of the RNAi machinery were not different from that in GC (e.g., Dicer, Argonaute, Piwi; P>0.05). The expression of 228 microRNAs, of the 232 analysed, were also unaffected during 4 and 8 d spaceflight (P>0.05). In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC. RNAi against rbx-1 also induced abnormal chromosome segregation in the gonad during spaceflight as on Earth. Finally, culture in RNAi against lysosomal cathepsins prevented degradation of the muscle-specific α-actin protein in both spaceflight and GC conditions.ConclusionsTreatment with RNAi works as effectively in the space environment as on Earth within multiple tissues, suggesting RNAi may provide an effective tool for combating spaceflight-induced pathologies aboard future long-duration space missions. Furthermore, this is the first demonstration that RNAi can be utilised to block muscle protein degradation, both on Earth and in space.

To estimate the space-radiation effects separately from other space-environmental effects such as microgravity, frozen human lymphoblastoid TK6 cells were sent to the "Kibo" module of the International Space Station (ISS), preserved under frozen condition during the mission and finally recovered to Earth (after a total of 134 days flight, 72 mSv). Biological assays were performed on the cells recovered to Earth. We observed a tendency of increase (2.3-fold) in thymidine kinase deficient (TK(-)) mutations over the ground control. Loss of heterozygosity (LOH) analysis on the mutants also demonstrated a tendency of increase in proportion of the large deletion (beyond the TK locus) events, 6/41 in the in-flight samples and 1/17 in the ground control. Furthermore, in-flight samples exhibited 48% of the ground-control level in TK(-) mutation frequency upon exposure to a subsequent 2 Gy dose of X-rays, suggesting a tendency of radioadaptation when compared with the ground-control samples. The tendency of radioadaptation was also supported by the post-flight assays on DNA double-strand break repair: a 1.8- and 1.7-fold higher efficiency of in-flight samples compared to ground control via non-homologous end-joining and homologous recombination, respectively. These observations suggest that this system can be used as a biodosimeter, because DNA damage generated by space radiation is considered to be accumulated in the cells preserved frozen during the mission, Furthermore, this system is also suggested to be applicable for evaluating various cellular responses to low-dose space radiation, providing a better understanding of biological space-radiation effects as well as estimation of health influences of future space explores.

The aim of this study was to determine the biological effects of space radiations, microgravity, and the interaction of them on the expression of p53-regulated proteins. Space experiments were performed with two human cultured lymphoblastoid cell lines: one line (TSCE5) bears a wild-type p53 gene status, and another line (WTK1) bears a mutated p53 gene status. Under 1 gravity or microgravity conditions, the cells were grown in the cell biology experimental facility (CBEF) of the International Space Station for 8 days without experiencing the stress during launching and landing because the cells were frozen during these periods. Ground control samples were simultaneously cultured for 8 days in the CBEF on the ground for 8 days. After spaceflight, protein expression was analyzed using a Panorama(TM) Ab MicroArray protein chips. It was found that p53-dependent up-regulated proteins in response to space radiations and space environment were MeCP2 (methyl CpG binding protein 2), and Notch1 (Notch homolog 1), respectively. On the other hand, p53-dependent down-regulated proteins were TGF-β, TWEAKR (tumor necrosis factor-like weak inducer of apoptosis receptor), phosho-Pyk2 (Proline-rich tyrosine kinase 2), and 14-3-3θ/τ which were affected by microgravity, and DR4 (death receptor 4), PRMT1 (protein arginine methyltransferase 1) and ROCK-2 (Rho-associated, coiled-coil containing protein kinase 2) in response to space radiations. ROCK-2 was also suppressed in response to the space environment. The data provides the p53-dependent regulated proteins by exposure to space radiations and/or microgravity during spaceflight. Our expression data revealed proteins that might help to advance the basic space radiation biology.