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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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Transfection of inhibitors decrease their corresponding endogenous miRNAs and transfection of mimics increase corresponding miRNAs in SHSY5Y cells.(A) SHSY5Y cells were transfected with hairpin inhibitors for human miR-200c, miR-182, miR-183 and miR-141 individually, the levels of these miRNAs were measured by qPCR, and expressed relative (fold change) to endogenous levels. (B) SHSY5Y cells were transfected with the double stranded miRNA mimics listed on the figure individually, the expression levels were measured by qPCR and the fold changes from endogenous levels were plotted.
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pone-0047787-g003: Transfection of inhibitors decrease their corresponding endogenous miRNAs and transfection of mimics increase corresponding miRNAs in SHSY5Y cells.(A) SHSY5Y cells were transfected with hairpin inhibitors for human miR-200c, miR-182, miR-183 and miR-141 individually, the levels of these miRNAs were measured by qPCR, and expressed relative (fold change) to endogenous levels. (B) SHSY5Y cells were transfected with the double stranded miRNA mimics listed on the figure individually, the expression levels were measured by qPCR and the fold changes from endogenous levels were plotted.

Mentions: We then used either inhibitors or mimics that were specific to the miRNAs of interest in order to examine the effects (or roles) of these miRNAs in regulating global ULM conjugation levels. We used miRIDIAN miRNA Mimics (Dharmacon), which are double-stranded RNA oligonucleotides chemically enhanced to mimic endogenous mature miRNA function, and miRIDIAN miRNA hairpin inhibitors (Dharmacon), which are single-stranded oligonucleotides designed to inhibit endogenous mature miRNA functions. As shown in Figure 3, inhibitors lowered the levels of these miRNAs (4∼64-fold compared to negative control transfected) (A), and mimics increased corresponding miRNAs levels (2000∼262,000-fold above negative control transfectants) (B). Although an ‘inhibitor’ would generally be thought not to change the level of the miRNA, but to prevent its mechanisms of action instead, the Dharmacon ‘inhibitor’ acts as a sink and reduces the availability of the miRNA, so the detected level becomes low. In many of the miRNA inhibitor assays, the active miRNA concentrations were lowered to background levels limiting the accuracy of the fold-inhibition calculation. The order of endogenous levels of the microRNAs tested in SHSY5Y cells is miR-182>>miR-141≥miR-183>miR-200c. The endogenous level of miR-182 is much higher than those of other miRNAs tested, thus showing greater apparent inhibition by its specific inhibitor, but a reduced fold increase by mimics. Conversely, the endogenous level of miR-200c is very low, so the apparent fold inhibition is low, but the fold increase by its specific mimic is huge. We transfected SHSY5Y cells with these inhibitors or mimics, and two days later ULM proteins and ULM global conjugation levels in the cells were analyzed by Western blot. As shown in Figure 4, when these miRNA expressions were inhibited by their specific inhibitors, most of the ULM conjugations examined were indeed increased. Conversely, when these miRNAs were overexpressed by their specific mimics, the conjugation levels of these ULMs were depressed. In addition, the expression of some of the free (unconjugated) ULMs (NEDD8 or UFM1) and the SUMO E2 conjugase, Ubc9, were also controlled by these miRNAs.


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)

Transfection of inhibitors decrease their corresponding endogenous miRNAs and transfection of mimics increase corresponding miRNAs in SHSY5Y cells.(A) SHSY5Y cells were transfected with hairpin inhibitors for human miR-200c, miR-182, miR-183 and miR-141 individually, the levels of these miRNAs were measured by qPCR, and expressed relative (fold change) to endogenous levels. (B) SHSY5Y cells were transfected with the double stranded miRNA mimics listed on the figure individually, the expression levels were measured by qPCR and the fold changes from endogenous levels were plotted.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3475703&req=5

pone-0047787-g003: Transfection of inhibitors decrease their corresponding endogenous miRNAs and transfection of mimics increase corresponding miRNAs in SHSY5Y cells.(A) SHSY5Y cells were transfected with hairpin inhibitors for human miR-200c, miR-182, miR-183 and miR-141 individually, the levels of these miRNAs were measured by qPCR, and expressed relative (fold change) to endogenous levels. (B) SHSY5Y cells were transfected with the double stranded miRNA mimics listed on the figure individually, the expression levels were measured by qPCR and the fold changes from endogenous levels were plotted.
Mentions: We then used either inhibitors or mimics that were specific to the miRNAs of interest in order to examine the effects (or roles) of these miRNAs in regulating global ULM conjugation levels. We used miRIDIAN miRNA Mimics (Dharmacon), which are double-stranded RNA oligonucleotides chemically enhanced to mimic endogenous mature miRNA function, and miRIDIAN miRNA hairpin inhibitors (Dharmacon), which are single-stranded oligonucleotides designed to inhibit endogenous mature miRNA functions. As shown in Figure 3, inhibitors lowered the levels of these miRNAs (4∼64-fold compared to negative control transfected) (A), and mimics increased corresponding miRNAs levels (2000∼262,000-fold above negative control transfectants) (B). Although an ‘inhibitor’ would generally be thought not to change the level of the miRNA, but to prevent its mechanisms of action instead, the Dharmacon ‘inhibitor’ acts as a sink and reduces the availability of the miRNA, so the detected level becomes low. In many of the miRNA inhibitor assays, the active miRNA concentrations were lowered to background levels limiting the accuracy of the fold-inhibition calculation. The order of endogenous levels of the microRNAs tested in SHSY5Y cells is miR-182>>miR-141≥miR-183>miR-200c. The endogenous level of miR-182 is much higher than those of other miRNAs tested, thus showing greater apparent inhibition by its specific inhibitor, but a reduced fold increase by mimics. Conversely, the endogenous level of miR-200c is very low, so the apparent fold inhibition is low, but the fold increase by its specific mimic is huge. We transfected SHSY5Y cells with these inhibitors or mimics, and two days later ULM proteins and ULM global conjugation levels in the cells were analyzed by Western blot. As shown in Figure 4, when these miRNA expressions were inhibited by their specific inhibitors, most of the ULM conjugations examined were indeed increased. Conversely, when these miRNAs were overexpressed by their specific mimics, the conjugation levels of these ULMs were depressed. In addition, the expression of some of the free (unconjugated) ULMs (NEDD8 or UFM1) and the SUMO E2 conjugase, Ubc9, were also controlled by these miRNAs.

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