The Effectiveness of RNAi in Caenorhabditis elegans Is Maintained during Spaceflight

PublishedJournal ArticleResearch Support, N.I.H., ExtramuralResearch Support, Non-U.S. Gov'tThis is the final version of the article. Available from Public Library of Science via the DOI in this record.BACKGROUND: Overcoming 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. RESULTS: After 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. CONCLUSIONS: Treatment 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.This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Japan Society for the Promotion of Science, and “Ground-Based Research Announcement for Space Utilization” promoted by the Japan Space Forum. TE was supported by the Medical Research Council UK (G0801271). NJS was supported by the National Institutes of Health (NIH NIAMS ARO54342). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Spaceflight results in increase of thick filament but not thin filament proteins in the paramyosin mutant of Caenorhabditis elegans

We have investigated the effect of microgravity during spaceflight on body-wall muscle fiber size and muscle proteins in the paramyosin mutant of Caenorhabditis elegans. Both mutant and wild-type strains were subjected to 10 days of microgravity during spaceflight and compared to ground control groups. No significant change in muscle fiber size or quantity of the protein was observed in wild-type worms; where as atrophy of body-wall muscle and an increase in thick filament proteins were observed in the paramyosin mutant unc-15(e73) animals after spaceflight. We conclude that the mutant with abnormal muscle responded to microgravity by increasing the total amount of muscle protein in order to compensate for the loss of muscle function

Calpains Mediate Integrin Attachment Complex Maintenance of Adult Muscle in Caenorhabditis elegans

Two components of integrin containing attachment complexes, UNC-97/PINCH and UNC-112/MIG-2/Kindlin-2, were recently identified as negative regulators of muscle protein degradation and as having decreased mRNA levels in response to spaceflight. Integrin complexes transmit force between the inside and outside of muscle cells and signal changes in muscle size in response to force and, perhaps, disuse. We therefore investigated the effects of acute decreases in expression of the genes encoding these multi-protein complexes. We find that in fully developed adult Caenorhabditis elegans muscle, RNAi against genes encoding core, and peripheral, members of these complexes induces protein degradation, myofibrillar and mitochondrial dystrophies, and a movement defect. Genetic disruption of Z-line– or M-line–specific complex members is sufficient to induce these defects. We confirmed that defects occur in temperature-sensitive mutants for two of the genes: unc-52, which encodes the extra-cellular ligand Perlecan, and unc-112, which encodes the intracellular component Kindlin-2. These results demonstrate that integrin containing attachment complexes, as a whole, are required for proper maintenance of adult muscle. These defects, and collapse of arrayed attachment complexes into ball like structures, are blocked when DIM-1 levels are reduced. Degradation is also blocked by RNAi or drugs targeting calpains, implying that disruption of integrin containing complexes results in calpain activation. In wild-type animals, either during development or in adults, RNAi against calpain genes results in integrin muscle attachment disruptions and consequent sub-cellular defects. These results demonstrate that calpains are required for proper assembly and maintenance of integrin attachment complexes. Taken together our data provide in vivo evidence that a calpain-based molecular repair mechanism exists for dealing with attachment complex disruption in adult muscle. Since C. elegans lacks satellite cells, this mechanism is intrinsic to the muscles and raises the question if such a mechanism also exists in higher metazoans

Metabolism of halophilic archaea

In spite of their common hypersaline environment, halophilic archaea are surprisingly different in their nutritional demands and metabolic pathways. The metabolic diversity of halophilic archaea was investigated at the genomic level through systematic metabolic reconstruction and comparative analysis of four completely sequenced species: Halobacterium salinarum, Haloarcula marismortui, Haloquadratum walsbyi, and the haloalkaliphile Natronomonas pharaonis. The comparative study reveals different sets of enzyme genes amongst halophilic archaea, e.g. in glycerol degradation, pentose metabolism, and folate synthesis. The carefully assessed metabolic data represent a reliable resource for future system biology approaches as it also links to current experimental data on (halo)archaea from the literature

Fluid dynamics alter Caenorhabditis elegans body length via neuromuscular signaling with TGF-β/DBL-1

This is the author accepted manuscript. The final version is available from Nature Publishing Group via the DOI in this record.Skeletal muscle wasting is a major obstacle for long-term space exploration. Similar to astronauts, the nematode Caenorhabditis elegans displays negative muscular and physical effects grown in space microgravity. However, it is still unclear what signal molecules and behavior affect the negative alterations. We here studied key signaling molecules involved in alterations of C. elegans physique in response to fluid-dynamics on the ground-based experiments. Like as spaceflight experiment with 1G accelerator onboard, a myosin heavy chain myo-3 and a TGF- dbl-1 gene expression altered increasing the fluid dynamic parameters viscosity/drag resistance or depth of liquid culture. These gene expression also drastically increased grown liquid medium as compared with moist agar surface. In addition, body length enhanced in WT and body-wall cuticle collagen mutants, rol-6 roller and dpy-5 dumpy, grown in liquid culture. On the other hand, in a TGF- gene dbl-1 and its signaling pathway sma-4/Smad mutants, their body lengths did not alter in liquid. Similarly, a D1-like dopamine receptor DOP-4 and a mechanosensory channel UNC-8 were required for altered physique in which DBL-1 signaling did not upregulated in liquid. Since C. elegans contraction rates are much higher in swimming mode in liquid than clawing mode on agar surface, we studied the relationship between body-length enhancement and contraction rate. Mutants significantly reduced contraction rate commonly show smaller size, although the rate in dop-4, dbl-1 and sma-4 mutants still increased in liquid. These results suggest that neuromuscular signaling via TGF-/DBL-1 to alter body physique in response to environmental conditions including fluid dynamics.We are grateful to the entire crew of the CERISE for their work on STS-129, STS-130, and the International Space Station. The CERISE was organized with the support of the JAXA. We also thank the Caenorhabditis elegans Genetic Center for kindly supplying the mutant strains. This work was also supported by JSPS KAKENHI grant numbers 26506029, 15H05937, the Cross-ministerial Strategic Innovation Promotion Program (J150000592), the Medical Research Council UK (G0801271), and National Institutes of Health (NIH NIAMS ARO54342). This work was supported by grants from the MEXT, the JSPS (15H05937, 26506029), the Cross-ministerial Strategic Innovation Promotion Program (J150000592), and the Cell Biology Experiment Project conducted by the Institute of Space and Astronautical Science in JAXA. TE was supported by the Medical Research Council of UK (G0801271). NJS was supported by the National Institutes of Health (NIH NIAMS ARO54342)

Microgravity elicits reproducible alterations in cytoskeletal and metabolic gene and protein expression in space-flown Caenorhabditis elegans

This is the author accepted manuscript. The final version is available from Nature Publishing Group via http://dx.doi.org/10.1038/npjmgrav.2015.22Although 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 are grateful to the entire crew of the CERISE for their work on STS-129, STS-130, and the ISS. The CERISE was organized with the support of the JAXA. This experiment was supported by the Cell Biology Experiment Project conducted by the Institute of Space and Astronautical Science in JAXA, and was funded in part by JSPS KAKENHI Grant Numbers 26506029, 15H05937, the Medical Research Council UK (G0801271), and National Institutes of Health (NIH NIAMS ARO54342)