Elevated Global SUMOylation in Ubc9 Transgenic Mice Protects Their Brains against Focal Cerebral Ischemic Damage

We have previously shown that a massive increase in global SUMOylation occurs during torpor in ground squirrels, and that overexpression of Ubc9 and/or SUMO-1 in cell lines and cortical neurons protects against oxygen and glucose deprivation. To examine whether increased global SUMOylation protects against ischemic brain damage, we have generated transgenic mice in which Ubc9 is expressed strongly in all tissues under the chicken β-actin promoter. Ubc9 expression levels in 10 founder lines ranged from 2 to 30 times the endogenous level, and lines that expressed Ubc9 at modestly increased levels showed robust resistance to brain ischemia compared to wild type mice. The infarction size was inversely correlated with the Ubc9 expression levels for up to five times the endogenous level. Although further increases showed no additional benefit, the Ubc9 expression level was highly correlated with global SUMO-1 conjugation levels (and SUMO-2,3 levels to a lesser extent) up to a five-fold Ubc9 increase. Most importantly, there were striking reciprocal relationships between SUMO-1 (and SUMO-2,3) conjugation levels and cerebral infarction volumes among all tested animals, suggesting that the limit in cytoprotection by global SUMOylation remains undefined. These results support efforts to further augment global protein SUMOylation in brain ischemia

Akt Protein Kinase, miR-200/miR-182 Expression and Epithelial-Mesenchymal Transition Proteins in Hibernating Ground Squirrels

Hibernating 13-lined ground squirrels (Ictidomys tridecemlineatus; TLGS) rank among the most brain hypoperfusion-tolerant mammals known. Herein we provide some evidence of cycling between an epithelial phenotype and a hybrid epithelial/mesenchymal (E/M) phenotype (partial EMT) within the brains of TLGS during each bout of hibernation torpor. During hibernation torpor, expression of the epithelial marker E-cadherin (E-CDH) was reduced, while expression of the well-known mesenchymal markers vimentin and Sox2 were increased. P-cadherin (P-CDH), which has recently been proposed as a marker of intermediate/partial EMT, also increased during torpor, suggesting that a partial EMT may be taking place during hibernation torpor. Members of the miR-200 family and miR-182 cluster and Akt isoforms (Akt1, Akt2), well-known EMT regulators, were also differentially regulated in the TLGS brain during hibernation bouts. Using SHSY5Y cells, we also demonstrate that the Akt1/Akt2 ratio determined the expression levels of miR-200/miR-182 miRNA family members, and that these miRNAs controlled the expression of EMT-related proteins. Accordingly, we propose that such cell state transitions (EMT/MET) may be one of the mechanisms underlying the extraordinary ischemic tolerance of the TLGS brain during hibernation bouts; hibernator brain cells appear to enter reversible states that confer the stress survival characteristics of cancer cells without the risk of neoplastic transformation

Synthetic Oligodeoxynucleotides Containing Multiple Telemeric TTAGGG Motifs Suppress Inflammasome Activity in Macrophages Subjected to Oxygen and Glucose Deprivation and Reduce Ischemic Brain Injury in Stroke-Prone Spontaneously Hypertensive Rats.

The immune system plays a fundamental role in both the development and pathobiology of stroke. Inflammasomes are multiprotein complexes that have come to be recognized as critical players in the inflammation that ultimately contributes to stroke severity. Inflammasomes recognize microbial and host-derived danger signals and activate caspase-1, which in turn controls the production of the pro-inflammatory cytokine IL-1β. We have shown that A151, a synthetic oligodeoxynucleotide containing multiple telemeric TTAGGG motifs, reduces IL-1β production by activated bone marrow derived macrophages that have been subjected to oxygen-glucose deprivation and LPS stimulation. Further, we demonstrate that A151 reduces the maturation of caspase-1 and IL-1β, the levels of both the iNOS and NLRP3 proteins, and the depolarization of mitochondrial membrane potential within such cells. In addition, we have demonstrated that A151 reduces ischemic brain damage and NLRP3 mRNA levels in SHR-SP rats that have undergone permanent middle cerebral artery occlusion. These findings clearly suggest that the modulation of inflammasome activity via A151 may contribute to a reduction in pro-inflammatory cytokine production by macrophages subjected to conditions that model brain ischemia and modulate ischemic brain damage in an animal model of stroke. Therefore, modulation of ischemic pathobiology by A151 may have a role in the development of novel stroke prevention and therapeutic strategies

A151 reduced depolarization of mitochondrial membrane potential (MMP) in BMDM subjected to LPS and OGD.

<p>(A) FACS analysis of cells stained with JC-1. (B) The percentage of cells with depolarized MMP was reduced by A151 treatment. Data are presented as mean ± SEM from three replicates representative of three independent experiments (**, <i>p</i> < 0.05 compared with control or C151 treatment).</p

A151 reduced brain ischemic injury in SHR-SP rats 48 hours after pMCAO.

<p>The rats in saline groups were combined for analysis, as they were not statistically different. (A) Representative coronal brain sections stained with cresyl violet. (B) A151 reduced infarct volumes in male rats. (C) A151 reduced infarct volumes in female rats. (D) A151 improved performance in forepaw test in female rats. (E) A151 reduced brain NLRP3 mRNA 48 hours after pMCAO, the error bars represent the 95^th upper and lower confidence intervals of gene expression. (n = 7–17 per group; *, <i>p</i> < 0.05 compared with saline control; **, <i>p</i> < 0.05 compared with saline control or C151).</p

A151 reduced pro-inflammatory cytokine production and cell death in BMDM subjected to LPS and OGD.

<p>BMDM were treated with OGD, with or without 1 ng/ml LPS, A151 or C151 for 18 hours. IL-1β (A), IL-1α (B), IL-6 (C), CINC-1 (D), TNFα (E), and LDH (F) in cell culture supernatants were measured by ELISA. Data are presented as mean ± SEM from three replicates representative of three independent experiments (*, <i>p</i> < 0.05 compared with LPS treatment; **, <i>p</i> < 0.05 compared with control or C151 treatment).</p

A151 reduced IL-1β and caspase 1 maturation, and the expression of NLRP3 and iNOS in BMDM subjected to LPS and OGD.

<p>(A) A151 reduced mature IL-1β in supernatants. (B) A151 reduced mature caspase 1 in supernatants. (C) A151 did not influence caspase 11 in cell lysates or supernatants. (D) A151 reduced NLRP3 in cell lysates. (E) A151 reduced iNOS in cell lysates. Data are presented as mean ± SEM from three replicates representative of three independent experiments (**, <i>p</i> < 0.05 compared with control or C151 treatment).</p