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

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.

Figure 1. Protein Conjugation by various ULMs increases during hibernation torpor in the brains of 13-lined ground squirrels.

(A) Representative Immunoblots of various ULMs in the brain samples from different stages of hibernation cycle. Arrow in each panel shows the molecular weight range of conjugates of the ULM indicated on top of the panel. a: ACR (active in the cold room); b: Ent (entrance); c: E-hib (early hibernation); d: L-hib (late hibernation); e:AR (arousal); f: IB (interabout). β-actin served as a loading control. Each immunoblot is representative of at least three different brain samples at each time point. (B) Quantitative analyses of the conjugates of various ULMs from three independent experiments. Cropped areas are shown by arrows; densities were measured, normalized by corresponding β-actin level, and shown as the ratio to ACR samples. Data represent the mean±SD of three independent experiments. **p<0.01, *p<0.05 compared to ACR.

Figure 2. 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.

Figure 3. 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.

Figure 4. Effect of miRNA inhibitors and mimics for miR-200 family and miR-182 family members on ULM conjugation levels in SHSY5Y cells.

(A) Representative Immunoblots of various ULMs in SHSY5Y cells that had been transfected with either miR inhibitors (left column) or mimics (right column) in each panel. (B) Quantitative analyses of the conjugates (left panel) or free form (right panel) of various ULMs in inhibitor-transfected SHSY5Y. (C) Quantitive analyses of the conjugates (left panel) or free form (right panel) of various ULMs in mimic-transfected SHSY5Y cells. Densities of conjugates or free forms were measured, normalized by corresponding β-actin level, and shown as the ratio to endogenous (negative control-transfected) level. Data represent the mean±SD of three independent experiments. **p<0.01, *p<0.05 compared with endogenous level.

Figure 5. miR-200 family and miR-182 family members of miRNAs indeed target various ULM and/or ULM related genes.

SHSY5Y cells were co-transfected with the pmirGLO Dual-Luciferase miRNA Target Expression (Promega) constructs into which putative target sites for the various ULMs had been inserted, and with mimics of miRNAs as indicated. Two days later, cells were lysed, luciferase acivities (both firefly and Renilla) were measured, firefly luciferase activities were normalized with Renilla firefly activity, and expressed as relative to control (transfected with empty vector and negative control mimic). Data are shown as the mean±SD of three independent experiments. ***p<0.001, **p<0.01, *p<0.05 compared to control.

Figure 6. 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.