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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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Effects of inhibitors and mimics for miR-200 family and miR-182 family miRNAs on OGD-induced cell death in SHSY5Y.(A) SHSY5Y cells were transfected with either inhibitors or mimics as indicated and 2days later the cell viability was measured by WST-1. Viability was expressed as percentage of negative control for either inhibitor or mimic transfected cells. (B) SHSY5Y cells transfected as mentioned above, and 2days later these cells were subjected to OGD (16 h), and viable cells were measured by WST-1. Viability was expressed as percentage of negative control cells (negative control inhibitor or negative control mimic transfected and not subjected to OGD). In both A and B, inhibitor transfected cells were shown in blue, and mimic transfected cells were shown in red. Data are shown as the mean±SD of three independent experiments. **p<0.01, *p<0.05 compared to control.
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pone-0047787-g006: Effects of inhibitors and mimics for miR-200 family and miR-182 family miRNAs on OGD-induced cell death in SHSY5Y.(A) SHSY5Y cells were transfected with either inhibitors or mimics as indicated and 2days later the cell viability was measured by WST-1. Viability was expressed as percentage of negative control for either inhibitor or mimic transfected cells. (B) SHSY5Y cells transfected as mentioned above, and 2days later these cells were subjected to OGD (16 h), and viable cells were measured by WST-1. Viability was expressed as percentage of negative control cells (negative control inhibitor or negative control mimic transfected and not subjected to OGD). In both A and B, inhibitor transfected cells were shown in blue, and mimic transfected cells were shown in red. Data are shown as the mean±SD of three independent experiments. **p<0.01, *p<0.05 compared to control.

Mentions: We have established that the miR-200 family and the miR-182 family miRNAs target SUMO and other ULMs, and that inactivation of these miRNAs increases both global SUMOylation and global conjugation of other ULMs resembling the changes in posttranslational modifications that have been noted in ground squirrels during hibernation torpor. Conversely, overexpression of these miRNAs by introducing their mimics caused a decrease in ULM conjugation levels. We wondered whether the tolerance to in vitro “ischemia” by OGD in cultured cells (e.g. SHSY5Y) would change if miR-200 family and/or miR-182 family miRNA activity levels were reduced or increased. We transfected SHSY5Y cells with inhibitors or mimics for several miR-200 family and the miR-182 family miRNAs (miR-200c, miR-141, miR-182, miR-183 and miR-96) in duplicate plates, incubated them for 2 days, and subjected one plate of cells to OGD, and the other plate of cells to normal culture conditiions as a control. As shown in Fig. 6A, without OGD the viability of cells transfected with miRNA inhibitors equaled or exceeded that of negative control transfected cells, but cells transfected with miRNA mimics showed 10∼30% lower viability than the cells transfected with a negative control inhibitor or mimic construct. OGD overnight (16 h) caused 60∼70% cell death (30∼40% in viability) in negative control transfected cells (Fig. 6B). Cells transfected with miRNA inhibitors, especially miR-200 family (miR-200c and miR-141) showed much far less cell death (Fig. 6B), but cells transfected with miRNA mimics, especially from the miR-182 family, caused more cell death (Fig. 6B). These results suggest that inhibiting miR-200 family and miR-182 family miRNAs does protect SHSY5Y cells from OGD-induced cell death. In the case of mimics, however, overexpressing these miRNAs alone caused some cell death without OGD, so the cell death after OGD might not be completely reflective of OGD-caused cell death.


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)

Effects of inhibitors and mimics for miR-200 family and miR-182 family miRNAs on OGD-induced cell death in SHSY5Y.(A) SHSY5Y cells were transfected with either inhibitors or mimics as indicated and 2days later the cell viability was measured by WST-1. Viability was expressed as percentage of negative control for either inhibitor or mimic transfected cells. (B) SHSY5Y cells transfected as mentioned above, and 2days later these cells were subjected to OGD (16 h), and viable cells were measured by WST-1. Viability was expressed as percentage of negative control cells (negative control inhibitor or negative control mimic transfected and not subjected to OGD). In both A and B, inhibitor transfected cells were shown in blue, and mimic transfected cells were shown in red. Data are shown as the mean±SD of three independent experiments. **p<0.01, *p<0.05 compared to control.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3475703&req=5

pone-0047787-g006: Effects of inhibitors and mimics for miR-200 family and miR-182 family miRNAs on OGD-induced cell death in SHSY5Y.(A) SHSY5Y cells were transfected with either inhibitors or mimics as indicated and 2days later the cell viability was measured by WST-1. Viability was expressed as percentage of negative control for either inhibitor or mimic transfected cells. (B) SHSY5Y cells transfected as mentioned above, and 2days later these cells were subjected to OGD (16 h), and viable cells were measured by WST-1. Viability was expressed as percentage of negative control cells (negative control inhibitor or negative control mimic transfected and not subjected to OGD). In both A and B, inhibitor transfected cells were shown in blue, and mimic transfected cells were shown in red. Data are shown as the mean±SD of three independent experiments. **p<0.01, *p<0.05 compared to control.
Mentions: We have established that the miR-200 family and the miR-182 family miRNAs target SUMO and other ULMs, and that inactivation of these miRNAs increases both global SUMOylation and global conjugation of other ULMs resembling the changes in posttranslational modifications that have been noted in ground squirrels during hibernation torpor. Conversely, overexpression of these miRNAs by introducing their mimics caused a decrease in ULM conjugation levels. We wondered whether the tolerance to in vitro “ischemia” by OGD in cultured cells (e.g. SHSY5Y) would change if miR-200 family and/or miR-182 family miRNA activity levels were reduced or increased. We transfected SHSY5Y cells with inhibitors or mimics for several miR-200 family and the miR-182 family miRNAs (miR-200c, miR-141, miR-182, miR-183 and miR-96) in duplicate plates, incubated them for 2 days, and subjected one plate of cells to OGD, and the other plate of cells to normal culture conditiions as a control. As shown in Fig. 6A, without OGD the viability of cells transfected with miRNA inhibitors equaled or exceeded that of negative control transfected cells, but cells transfected with miRNA mimics showed 10∼30% lower viability than the cells transfected with a negative control inhibitor or mimic construct. OGD overnight (16 h) caused 60∼70% cell death (30∼40% in viability) in negative control transfected cells (Fig. 6B). Cells transfected with miRNA inhibitors, especially miR-200 family (miR-200c and miR-141) showed much far less cell death (Fig. 6B), but cells transfected with miRNA mimics, especially from the miR-182 family, caused more cell death (Fig. 6B). These results suggest that inhibiting miR-200 family and miR-182 family miRNAs does protect SHSY5Y cells from OGD-induced cell death. In the case of mimics, however, overexpressing these miRNAs alone caused some cell death without OGD, so the cell death after OGD might not be completely reflective of OGD-caused cell death.

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