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Global protein conjugation by ubiquitin-like-modifiers during ischemic stress is regulated by microRNAs and confers robust tolerance to ischemia.

Lee YJ, Johnson KR, Hallenbeck JM - PLoS ONE (2012)

Bottom Line: We found that not only SUMO conjugation, but also global protein conjugation by other ULMs including NEDD8, ISG15, UFM1 and FUB1 were significantly increased in the brains of hibernating ground squirrels during torpor.By means of miRNA microarrays of ground squirrel brain samples (from active and torpor phase) we found that the miR-200 family (miR-200a,b,c/miR-141/miR-429) and the miR-182 family (miR-182/miR-183/miR-96) were among the most consistently depressed miRNAs in the brain during the torpor phase as compared to active animals.This is the first report to describe that the natural tolerance to brain ischemia in hibernators is linked to regulation by microRNAs of a broad range of ubiquitin-like modifiers.

View Article: PubMed Central - PubMed

Affiliation: Stroke Branch, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.

ABSTRACT
Hibernation torpor provides an excellent model of natural tolerance to ischemia. We have previously shown that massive global SUMOylation occurs during hibernation torpor in ground squirrels. We have also shown that overexpression of Ubc9, SUMO-1, or SUMO-2/3 provides protection against ischemic damage in cell lines and cortical neurons exposed to oxygen/glucose deprivation, and in mice exposed to middle cerebral artery occlusion. We have now extended our study to other Ubiquitin-Like-Modifiers (ULMs), which have multiple cellular functions during stress, in order to assess the possibility that they also have roles in tolerance to ischemia. We found that not only SUMO conjugation, but also global protein conjugation by other ULMs including NEDD8, ISG15, UFM1 and FUB1 were significantly increased in the brains of hibernating ground squirrels during torpor. By means of miRNA microarrays of ground squirrel brain samples (from active and torpor phase) we found that the miR-200 family (miR-200a,b,c/miR-141/miR-429) and the miR-182 family (miR-182/miR-183/miR-96) were among the most consistently depressed miRNAs in the brain during the torpor phase as compared to active animals. In addition, we showed that these miRNAs are involved in the expression of various ULM proteins and their global conjugation to proteins. We observed that inhibition of the miR-200 family and/or miR-182 family miRNA activities in SHSY5Y cells increases global protein conjugation by the above ULMs and makes these cells more tolerant to OGD-induced cell death. This is the first report to describe that the natural tolerance to brain ischemia in hibernators is linked to regulation by microRNAs of a broad range of ubiquitin-like modifiers.

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Differentially regulated miRNA families in the brains of 13-lined ground squirrels during hibernation.(A) 405 miRNAs which had an absolute mean difference between LH and ACR≥1.25 (using 6 different animal samples for each condition) were grouped into 33 families, and plotted against the frequencies observed. Down-regulated miRNAs were shown in red, and up-regulated miRNAs were shown in green. The number on top of each column is the fold change (ACR vs LH). (negative number = decrease, positive number = increase). (B) A subset of differentially regulated miRNAs detected by miRNA microarray was validated by qPCR using the same RNA samples that had been used for the miRNA microarray. The levels of the miRNAs were normalized by the level of miR-103, which was among the most stably expressed miRNAs with expression levels that did not change during the hibernation bout, and expressed as ratios of LH/ACR.
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pone-0047787-g002: Differentially regulated miRNA families in the brains of 13-lined ground squirrels during hibernation.(A) 405 miRNAs which had an absolute mean difference between LH and ACR≥1.25 (using 6 different animal samples for each condition) were grouped into 33 families, and plotted against the frequencies observed. Down-regulated miRNAs were shown in red, and up-regulated miRNAs were shown in green. The number on top of each column is the fold change (ACR vs LH). (negative number = decrease, positive number = increase). (B) A subset of differentially regulated miRNAs detected by miRNA microarray was validated by qPCR using the same RNA samples that had been used for the miRNA microarray. The levels of the miRNAs were normalized by the level of miR-103, which was among the most stably expressed miRNAs with expression levels that did not change during the hibernation bout, and expressed as ratios of LH/ACR.

Mentions: MicroRNAs are small noncoding RNAs that regulate a broad spectrum of genes post-transcriptionally particularly under stress conditions [8]. We ran microRNA arrays on brain samples from active and torpor phase ground squirrels (6 samples from each condition). 20,180 miRNA expression measurements representing 131 different organisms were generated per sample. No samples were removed as outliers. Measurements for 1,154 miRNAs were excluded as non-informative (miRNA not having at least one expression value across all samples greater than system noise were deemed non-informative), leaving 19,026 miRNA comparisons, of which 405 had an absolute mean difference in mean expression between LH and ACR≥1.25 (Table S1). For these 405 miRNAs, 33 families are represented (Fig. 2A). Of these families, miR-200 had the greatest number of differentially regulated members (n = 48) and the largest magnitude difference between means of the LH and ACR states (Δ = −3.04). Fold-differences for members within each family were found concordant 100% of the time with the exception of one family (miR-125)(Fig. 2A). In particular, two miRNA families, the miR-200 family (miR-200a, b, c, miR-496, and miR-141) and the miR-182 family (miR-182, miR-183 and miR-96) were consistently down regulated during torpor phase. In general, however, more miRNA families were upregulated during the torpor phase (Fig. 2A). We verified selected microRNA array data by reverse transcriptase (RT)-qPCR. We could confirm that miR-200, miR-182, miR-183, miR-141, miR-96, miR-122 and miR-429 were indeed down regulated, and miR-34 and miR-206 were upregulated during hibernation torpor (Fig. 2B). MiR-182, miR-96, miR-200, miR-183 and miR-141 were among the most highly regulated miRNAs with a 13∼20-fold decrease during the torpor phase of the hibernation cycle.


Global protein conjugation by ubiquitin-like-modifiers during ischemic stress is regulated by microRNAs and confers robust tolerance to ischemia.

Lee YJ, Johnson KR, Hallenbeck JM - PLoS ONE (2012)

Differentially regulated miRNA families in the brains of 13-lined ground squirrels during hibernation.(A) 405 miRNAs which had an absolute mean difference between LH and ACR≥1.25 (using 6 different animal samples for each condition) were grouped into 33 families, and plotted against the frequencies observed. Down-regulated miRNAs were shown in red, and up-regulated miRNAs were shown in green. The number on top of each column is the fold change (ACR vs LH). (negative number = decrease, positive number = increase). (B) A subset of differentially regulated miRNAs detected by miRNA microarray was validated by qPCR using the same RNA samples that had been used for the miRNA microarray. The levels of the miRNAs were normalized by the level of miR-103, which was among the most stably expressed miRNAs with expression levels that did not change during the hibernation bout, and expressed as ratios of LH/ACR.
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Related In: Results  -  Collection

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pone-0047787-g002: Differentially regulated miRNA families in the brains of 13-lined ground squirrels during hibernation.(A) 405 miRNAs which had an absolute mean difference between LH and ACR≥1.25 (using 6 different animal samples for each condition) were grouped into 33 families, and plotted against the frequencies observed. Down-regulated miRNAs were shown in red, and up-regulated miRNAs were shown in green. The number on top of each column is the fold change (ACR vs LH). (negative number = decrease, positive number = increase). (B) A subset of differentially regulated miRNAs detected by miRNA microarray was validated by qPCR using the same RNA samples that had been used for the miRNA microarray. The levels of the miRNAs were normalized by the level of miR-103, which was among the most stably expressed miRNAs with expression levels that did not change during the hibernation bout, and expressed as ratios of LH/ACR.
Mentions: MicroRNAs are small noncoding RNAs that regulate a broad spectrum of genes post-transcriptionally particularly under stress conditions [8]. We ran microRNA arrays on brain samples from active and torpor phase ground squirrels (6 samples from each condition). 20,180 miRNA expression measurements representing 131 different organisms were generated per sample. No samples were removed as outliers. Measurements for 1,154 miRNAs were excluded as non-informative (miRNA not having at least one expression value across all samples greater than system noise were deemed non-informative), leaving 19,026 miRNA comparisons, of which 405 had an absolute mean difference in mean expression between LH and ACR≥1.25 (Table S1). For these 405 miRNAs, 33 families are represented (Fig. 2A). Of these families, miR-200 had the greatest number of differentially regulated members (n = 48) and the largest magnitude difference between means of the LH and ACR states (Δ = −3.04). Fold-differences for members within each family were found concordant 100% of the time with the exception of one family (miR-125)(Fig. 2A). In particular, two miRNA families, the miR-200 family (miR-200a, b, c, miR-496, and miR-141) and the miR-182 family (miR-182, miR-183 and miR-96) were consistently down regulated during torpor phase. In general, however, more miRNA families were upregulated during the torpor phase (Fig. 2A). We verified selected microRNA array data by reverse transcriptase (RT)-qPCR. We could confirm that miR-200, miR-182, miR-183, miR-141, miR-96, miR-122 and miR-429 were indeed down regulated, and miR-34 and miR-206 were upregulated during hibernation torpor (Fig. 2B). MiR-182, miR-96, miR-200, miR-183 and miR-141 were among the most highly regulated miRNAs with a 13∼20-fold decrease during the torpor phase of the hibernation cycle.

Bottom Line: We found that not only SUMO conjugation, but also global protein conjugation by other ULMs including NEDD8, ISG15, UFM1 and FUB1 were significantly increased in the brains of hibernating ground squirrels during torpor.By means of miRNA microarrays of ground squirrel brain samples (from active and torpor phase) we found that the miR-200 family (miR-200a,b,c/miR-141/miR-429) and the miR-182 family (miR-182/miR-183/miR-96) were among the most consistently depressed miRNAs in the brain during the torpor phase as compared to active animals.This is the first report to describe that the natural tolerance to brain ischemia in hibernators is linked to regulation by microRNAs of a broad range of ubiquitin-like modifiers.

View Article: PubMed Central - PubMed

Affiliation: Stroke Branch, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, Maryland, United States of America.

ABSTRACT
Hibernation torpor provides an excellent model of natural tolerance to ischemia. We have previously shown that massive global SUMOylation occurs during hibernation torpor in ground squirrels. We have also shown that overexpression of Ubc9, SUMO-1, or SUMO-2/3 provides protection against ischemic damage in cell lines and cortical neurons exposed to oxygen/glucose deprivation, and in mice exposed to middle cerebral artery occlusion. We have now extended our study to other Ubiquitin-Like-Modifiers (ULMs), which have multiple cellular functions during stress, in order to assess the possibility that they also have roles in tolerance to ischemia. We found that not only SUMO conjugation, but also global protein conjugation by other ULMs including NEDD8, ISG15, UFM1 and FUB1 were significantly increased in the brains of hibernating ground squirrels during torpor. By means of miRNA microarrays of ground squirrel brain samples (from active and torpor phase) we found that the miR-200 family (miR-200a,b,c/miR-141/miR-429) and the miR-182 family (miR-182/miR-183/miR-96) were among the most consistently depressed miRNAs in the brain during the torpor phase as compared to active animals. In addition, we showed that these miRNAs are involved in the expression of various ULM proteins and their global conjugation to proteins. We observed that inhibition of the miR-200 family and/or miR-182 family miRNA activities in SHSY5Y cells increases global protein conjugation by the above ULMs and makes these cells more tolerant to OGD-induced cell death. This is the first report to describe that the natural tolerance to brain ischemia in hibernators is linked to regulation by microRNAs of a broad range of ubiquitin-like modifiers.

Show MeSH
Related in: MedlinePlus