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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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RNAi against gfp reduces chromosomal GFP expression                            in spaceflight and ground control (GC).Animals fed RNAi vector control for 4 d from L1 larvae developed into                            normal adults in GC and spaceflight conditions. These animals also                            displayed GFP expression in oocytes and embryos in GC and spaceflight.                            Animals fed gfp RNAi for 4 d also developed normally to                            adulthood in GC and spaceflight, and demonstrated a loss of GFP                            expression in both GC and spaceflight. Scale bars represent 50                            µm.
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pone-0020459-g001: RNAi against gfp reduces chromosomal GFP expression in spaceflight and ground control (GC).Animals fed RNAi vector control for 4 d from L1 larvae developed into normal adults in GC and spaceflight conditions. These animals also displayed GFP expression in oocytes and embryos in GC and spaceflight. Animals fed gfp RNAi for 4 d also developed normally to adulthood in GC and spaceflight, and demonstrated a loss of GFP expression in both GC and spaceflight. Scale bars represent 50 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)

RNAi against gfp reduces chromosomal GFP expression                            in spaceflight and ground control (GC).Animals fed RNAi vector control for 4 d from L1 larvae developed into                            normal adults in GC and spaceflight conditions. These animals also                            displayed GFP expression in oocytes and embryos in GC and spaceflight.                            Animals fed gfp RNAi for 4 d also developed normally to                            adulthood in GC and spaceflight, and demonstrated a loss of GFP                            expression in both GC and spaceflight. Scale bars represent 50                            µm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3105993&req=5

pone-0020459-g001: RNAi against gfp reduces chromosomal GFP expression in spaceflight and ground control (GC).Animals fed RNAi vector control for 4 d from L1 larvae developed into normal adults in GC and spaceflight conditions. These animals also displayed GFP expression in oocytes and embryos in GC and spaceflight. Animals fed gfp RNAi for 4 d also developed normally to adulthood in GC and spaceflight, and demonstrated a loss of GFP expression in both GC and spaceflight. Scale bars represent 50 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