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Micromanaging freeze tolerance: the biogenesis and regulation of neuroprotective microRNAs in frozen brains

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Abstract

When temperatures plummet below 0 °C, wood frogs (Rana sylvatica) can endure the freezing of up to ~ 65% of their body water in extracellular ice masses, displaying no measurable brain activity, no breathing, no movement, and a flat-lined heart. To aid survival, frogs retreat into a state of suspended animation characterized by global suppression of metabolic functions and reprioritization of energy usage to essential survival processes that is elicited, in part, by the regulatory controls of microRNAs. The present study is the first to investigate miRNA biogenesis and regulation in the brain of a freeze tolerant vertebrate. Indeed, proper brain function and adaptations to environmental stimuli play a crucial role in coordinating stress responses. Immunoblotting of miRNA biogenesis factors illustrated an overall reduction in the majority of these processing proteins suggesting a potential suppression of miRNA maturation over the freeze–thaw cycle. This was coupled with a large-scale RT-qPCR analysis of relative expression levels of 113 microRNA species in the brains of control, 24 h frozen, and 8 h thawed R. sylvatica. Of the 41 microRNAs differentially regulated during freezing and thawing, only two were significantly upregulated. Bioinformatic target enrichment of the downregulated miRNAs, performed at the low temperatures experienced during freezing and thawing, predicted their involvement in the potential activation of various neuroprotective processes such as synaptic signaling, intracellular signal transduction, and anoxia/ischemia injury protection. The predominantly downregulated microRNA fingerprint identified herein suggests a microRNA-mediated cryoprotective mechanism responsible for maintaining neuronal functions and facilitating successful whole brain freezing and thawing.

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Acknowledgements

This work was supported by a Discovery Grant (Grant #6793) from the Natural Sciences and Engineering Research Council (NSERC) of Canada. KBS holds the Canada Research Chair in Molecular Physiology and HH holds a NSERC Postgraduate scholarship.

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  1. Department of Biology and Institute of Biochemistry, Carleton University, 1125 Colonel By Drive, Ottawa, ON, K1S 5B6, Canada

    Hanane Hadj-Moussa & Kenneth B. Storey

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  1. Hanane Hadj-Moussa
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Correspondence to Kenneth B. Storey.

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18_2018_2821_MOESM1_ESM.xlsx

Primers used for analysis of miRNA expression in the brain of R. sylvatica, including miRNA-specific forward primers, universal reverse primer, and the stem-loop adapter for reverse-transcription. (XLSX 13 kb)

18_2018_2821_MOESM2_ESM.xlsx

Relative expression levels of 113 miRNA species examined in the brain of R. sylvatica. MicroRNA relative expression was evaluated by RT-qPCR of reverse-transcribed, polyadenylated transcripts. Data represent means of n = 3–4 biological replicates from different animals ± SEM. Relative expression of genes was calculated by standardizing against snord68 expression. Control values were adjusted to 1 and the 24 h frozen and 8 h thawed values were expressed relative to the control. Statistical testing used a one-wat ANOVA with a Dunnett's post hoc test, *p < 0.05, **p < 0.01, and ***p <0.001. (XLSX 18 kb)

18_2018_2821_MOESM3_ESM.xlsx

Identity of the significantly enriched biological processes, protein members, and miRNA species identified using MCL clustered protein networks and functional GO ANALYSIS of the miRNAs downregulated in 24 h frozen frog brain. (XLSX 17 kb)

18_2018_2821_MOESM4_ESM.xlsx

Identity of the significantly enriched biological processes, protein members, and miRNA species identified using MCL clustered protein networks and functional GO ANALYSIS of the miRNAs downregulated in 8 h thawed frog brain. (XLSX 22 kb)

18_2018_2821_MOESM5_ESM.jpg

Full cluster map of functional target enrichment and network clustering of the subset of miRNAs downregulated in the brains of 24 h frozen wood frogs. Downstream miRNA target prediction was performed at -2 °C using FINDTAR3. Protein–protein interactions of the downstream networks was performed using the STRING high-confidence filter on the X. tropicalis database. MCL clustering and visualization was performed on CYTOSCAPE and coupled with functional biological enrichment using GO ANALYSIS. Refer to Supplementary Table S3 for more information on individual clusters, proteins, and the targeting of individual miRNA species. (JPEG 351 kb)

18_2018_2821_MOESM6_ESM.jpg

Full cluster map of functional target enrichment and network clustering of the subset of miRNAs downregulated in the brains of 8 h thawed wood frogs. Downstream miRNA target prediction was performed at 5 °C using FINDTAR3. Protein–protein interactions of the downstream networks was performed using the STRING high-confidence filter on the X. tropicalis database. MCL clustering and visualization was performed on CYTOSCAPE and coupled with functional biological enrichment using GO ANALYSIS. Refer to Supplementary Table S4 for more information on individual clusters, proteins, and the targeting of individual miRNA species. (JPEG 302 kb)

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Hadj-Moussa, H., Storey, K.B. Micromanaging freeze tolerance: the biogenesis and regulation of neuroprotective microRNAs in frozen brains. Cell. Mol. Life Sci. 75, 3635–3647 (2018). https://doi.org/10.1007/s00018-018-2821-0

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