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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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rbx-1 RNAi induces abnormal chromosomal GFP                            localisation in spaceflight and ground control (GC).Adult animals fed RNAi vector control from L1 larvae for 4 d produced                            normal eggs in GC and spaceflight, and display normal embryonic                            chromosomal GFP localisation in GC and spaceflight. RNAi against                                rbx-1 for 4 d caused abnormal embryo development in                            GC and spaceflight, and induced irregular embryonic nuclear segregation                            and arrest of meiotic division in both GC and spaceflight. Scale bars                            represent 10 µm.
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pone-0020459-g002: rbx-1 RNAi induces abnormal chromosomal GFP localisation in spaceflight and ground control (GC).Adult animals fed RNAi vector control from L1 larvae for 4 d produced normal eggs in GC and spaceflight, and display normal embryonic chromosomal GFP localisation in GC and spaceflight. RNAi against rbx-1 for 4 d caused abnormal embryo development in GC and spaceflight, and induced irregular embryonic nuclear segregation and arrest of meiotic division in both GC and spaceflight. Scale bars represent 10 µm.

Mentions: To directly determine if RNAi functions normally in spaceflight, L1 larvae of the strain AZ212 (integrated array pAZ132; pie-1::GFP::histone H2B fusion, which express histone-tagged green fluorescent protein (GFP) in the nuclei of oocytes and embryos) were prepared as above. Upon arrival in space larvae were grown to adulthood by culturing under three conditions: gfp RNAi; rbx-1 RNAi, and; vector control for 4 d before freezing at −80°C. RNAi against gfp was chosen due to its use in the seminal demonstration of the efficacy of RNAi in C. elegans [1]. RNAi against rbx-1 was employed for its previous validation by the authors [23]. Fluorescent light microscopy on return to Earth demonstrated that in vector controls, GFP expression levels were comparable between 4 d spaceflight and ground controls (figure 1). RNAi against gfp resulted in decreased embryonic GFP expression that was not different between spaceflight and ground controls (figure 1). Furthermore, in both spaceflight and ground control conditions, RNAi against rbx-1 induced abnormal embryonic nuclear segregation and arrest of meiotic division observed by histone::GFP localisation (figure 2).


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)

rbx-1 RNAi induces abnormal chromosomal GFP                            localisation in spaceflight and ground control (GC).Adult animals fed RNAi vector control from L1 larvae for 4 d produced                            normal eggs in GC and spaceflight, and display normal embryonic                            chromosomal GFP localisation in GC and spaceflight. RNAi against                                rbx-1 for 4 d caused abnormal embryo development in                            GC and spaceflight, and induced irregular embryonic nuclear segregation                            and arrest of meiotic division in both GC and spaceflight. Scale bars                            represent 10 µm.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105993&req=5

pone-0020459-g002: rbx-1 RNAi induces abnormal chromosomal GFP localisation in spaceflight and ground control (GC).Adult animals fed RNAi vector control from L1 larvae for 4 d produced normal eggs in GC and spaceflight, and display normal embryonic chromosomal GFP localisation in GC and spaceflight. RNAi against rbx-1 for 4 d caused abnormal embryo development in GC and spaceflight, and induced irregular embryonic nuclear segregation and arrest of meiotic division in both GC and spaceflight. Scale bars represent 10 µm.
Mentions: To directly determine if RNAi functions normally in spaceflight, L1 larvae of the strain AZ212 (integrated array pAZ132; pie-1::GFP::histone H2B fusion, which express histone-tagged green fluorescent protein (GFP) in the nuclei of oocytes and embryos) were prepared as above. Upon arrival in space larvae were grown to adulthood by culturing under three conditions: gfp RNAi; rbx-1 RNAi, and; vector control for 4 d before freezing at −80°C. RNAi against gfp was chosen due to its use in the seminal demonstration of the efficacy of RNAi in C. elegans [1]. RNAi against rbx-1 was employed for its previous validation by the authors [23]. Fluorescent light microscopy on return to Earth demonstrated that in vector controls, GFP expression levels were comparable between 4 d spaceflight and ground controls (figure 1). RNAi against gfp resulted in decreased embryonic GFP expression that was not different between spaceflight and ground controls (figure 1). Furthermore, in both spaceflight and ground control conditions, RNAi against rbx-1 induced abnormal embryonic nuclear segregation and arrest of meiotic division observed by histone::GFP localisation (figure 2).

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