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The effectiveness of RNAi in Caenorhabditis elegans is maintained during spaceflight.

Etheridge T, Nemoto K, Hashizume T, Mori C, Sugimoto T, Suzuki H, Fukui K, Yamazaki T, Higashibata A, Szewczyk NJ, Higashitani A - PLoS ONE (2011)

Bottom Line: In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Physiology, Royal Derby Hospital, University of Nottingham, Derby, England.

ABSTRACT

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.

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Degradation of α-actin is prevented by asp-4 and                                asp-6 RNAi in spaceflight and ground control                            (GC).Dauer animals treated for 4 d with RNAi vector control (VC) developed to                            adulthood. In both GC and spaceflight conditions animals displayed major                            loss of muscle specific α-actin following lysis in the absence of                            lysosomal protease inhibitors. Treatment with asp-4 and                                asp-6 RNAi for 4 d in GC and spaceflight resulted                            in a preservation of α-actin levels. A, representative immunoblot;                            B, average non-normalised quantification of three Western blots against                            α-actin. ** denotes significant difference from both GC and                            spaceflight VC conditions (P<0.01).
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pone-0020459-g003: Degradation of α-actin is prevented by asp-4 and asp-6 RNAi in spaceflight and ground control (GC).Dauer animals treated for 4 d with RNAi vector control (VC) developed to adulthood. In both GC and spaceflight conditions animals displayed major loss of muscle specific α-actin following lysis in the absence of lysosomal protease inhibitors. Treatment with asp-4 and asp-6 RNAi for 4 d in GC and spaceflight resulted in a preservation of α-actin levels. A, representative immunoblot; B, average non-normalised quantification of three Western blots against α-actin. ** denotes significant difference from both GC and spaceflight VC conditions (P<0.01).

Mentions: Finally, to test whether RNAi against lysosomal cathepsins in space (asp-4, asp-6) prevented the degradation of muscle protein α-actin on return to Earth, dauer animals were flown in liquid media as above. On arrival in space dauers were cultured in either a vector control or asp-4 and asp-6 [24] RNAi for 4 d until adulthood. Samples were prepared for Western blot analysis in the presence of a protease inhibitor cocktail, which inhibits the activity of the proteasome, calpains and caspases but not that of lysosomal enzymes. Immunoblotting was performed against α-actin for its specificity to muscle, with β-actin used as a ubiquitously expressed loading control. Multiple Western blots (each blot using a different primary antibody against α-actin) revealed a preservation of α-actin protein levels in animals cultured in the presence of asp-4 and asp-6 RNAi versus vector control, in both spaceflight and ground control conditions. A near complete loss of α-actin, within limits of detection, was observed in animals cultured in vector control (figure 3); these observations were found to be statistically significant (P<0.01).


The effectiveness of RNAi in Caenorhabditis elegans is maintained during spaceflight.

Etheridge T, Nemoto K, Hashizume T, Mori C, Sugimoto T, Suzuki H, Fukui K, Yamazaki T, Higashibata A, Szewczyk NJ, Higashitani A - PLoS ONE (2011)

Degradation of α-actin is prevented by asp-4 and                                asp-6 RNAi in spaceflight and ground control                            (GC).Dauer animals treated for 4 d with RNAi vector control (VC) developed to                            adulthood. In both GC and spaceflight conditions animals displayed major                            loss of muscle specific α-actin following lysis in the absence of                            lysosomal protease inhibitors. Treatment with asp-4 and                                asp-6 RNAi for 4 d in GC and spaceflight resulted                            in a preservation of α-actin levels. A, representative immunoblot;                            B, average non-normalised quantification of three Western blots against                            α-actin. ** denotes significant difference from both GC and                            spaceflight VC conditions (P<0.01).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105993&req=5

pone-0020459-g003: Degradation of α-actin is prevented by asp-4 and asp-6 RNAi in spaceflight and ground control (GC).Dauer animals treated for 4 d with RNAi vector control (VC) developed to adulthood. In both GC and spaceflight conditions animals displayed major loss of muscle specific α-actin following lysis in the absence of lysosomal protease inhibitors. Treatment with asp-4 and asp-6 RNAi for 4 d in GC and spaceflight resulted in a preservation of α-actin levels. A, representative immunoblot; B, average non-normalised quantification of three Western blots against α-actin. ** denotes significant difference from both GC and spaceflight VC conditions (P<0.01).
Mentions: Finally, to test whether RNAi against lysosomal cathepsins in space (asp-4, asp-6) prevented the degradation of muscle protein α-actin on return to Earth, dauer animals were flown in liquid media as above. On arrival in space dauers were cultured in either a vector control or asp-4 and asp-6 [24] RNAi for 4 d until adulthood. Samples were prepared for Western blot analysis in the presence of a protease inhibitor cocktail, which inhibits the activity of the proteasome, calpains and caspases but not that of lysosomal enzymes. Immunoblotting was performed against α-actin for its specificity to muscle, with β-actin used as a ubiquitously expressed loading control. Multiple Western blots (each blot using a different primary antibody against α-actin) revealed a preservation of α-actin protein levels in animals cultured in the presence of asp-4 and asp-6 RNAi versus vector control, in both spaceflight and ground control conditions. A near complete loss of α-actin, within limits of detection, was observed in animals cultured in vector control (figure 3); these observations were found to be statistically significant (P<0.01).

Bottom Line: In spaceflight, RNAi against green fluorescent protein (gfp) reduced chromosomal gfp expression in gonad tissue, which was not different from GC.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.

View Article: PubMed Central - PubMed

Affiliation: Division of Clinical Physiology, Royal Derby Hospital, University of Nottingham, Derby, England.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus