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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.

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.

The molecular mechanisms underlying hypothermic neuroprotection have yet to be fully elucidated. Herein we demonstrate that global SUMOylation, a form of post-translational modification with the Small Ubiquitin-like MOdifer, participates in the multimodal molecular induction of hypothermia-induced ischemic tolerance. Mild (32°C) to moderate (28°C) hypothermic treatment(s) during OGD (oxygen-glucose-deprivation) or ROG (restoration of oxygen/glucose) increased global SUMO-conjugation levels and protected cells (both SHSY5Y and E18 rat cortical neurons) from OGD and ROG-induced cell death. Hypothermic exposure either before or after permanent middle cerebral artery occlusion (pMCAO) surgery in wild type mice increased global SUMO-conjugation levels in the brain and in so doing protected these animals from pMCAO-induced ischemic damage. Of note, hypothermic exposure did not provide an additional increase in protection from pMCAO-induced ischemic brain damage in Ubc9 transgenic (Ubc9 Tg) mice, which overexpress the sole E2 SUMO conjugating enzyme and thereby display elevated basal levels of global SUMOylation under normothermic conditions. Such evidence suggests that increases in global SUMOylation are critical and may account for a substantial part of the observed increase in cellular tolerance to brain ischemia caused via hypothermia.

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.

The serine-threonine protein kinase Akt has been identified as an important mediator of cell survival able to counteract apoptotic stimuli. However, hibernation, a model of natural tolerance to cerebral ischemia, is associated with downregulation of Akt. We previously established a model of ischemic tolerance in a PC12 cell line and using this model we now addressed the question whether ischemic tolerance also downregulates Akt in PC12 cells. Kinetic studies showed decreased Akt phosphorylation in tolerized cells. Similarly, phosphorylated levels of three major targets of Akt and well-known proapoptotic factors, the glycogen synthase kinase 3 (GSK-3), a Forkhead family member, FoxO4, and the protein murine double minute 2 (MDM2), all inactivated upon phosphorylation by Akt, were decreased in preconditioned cells. In addition, pharmacological blockade of the phosphoinositide 3-kinase (PI3K)/Akt pathway reduced cell death induced by oxygen and glucose deprivation (OGD) and increased the protective effect of preconditioning (PC). Furthermore, decreasing availability of P-Akt by transfecting PC12 cells with constructs of inactive Akt also resulted in protection against OGD and potentiation of the protective effect of PC. Depending on the environment, GSK-3, FOXO-4, and MDM2 can trigger apoptotic responses or cell cycle arrest, and thus, in a situation of reduced energy, driving the cells into a state of quiescence might be neuroprotective. This work suggests that in the context of tolerance downregulation of Akt is beneficial.

After 24-hour middle cerebral artery occlusion (MCAO) in spontaneously hypertensive rats, brain ceramide level increased from baseline reached 595% (ischemic core) and 460% (perifocal/penumbral areas); brain glucosylceramide synthase (GCS) activities in these areas simultaneously decreased by 70% and 50%, respectively. Ten-minute MCAO preconditioning significantly attenuated 24-hour MCAO-induced ceramide accumulation by 40% to 60% in ischemic core and perifocal areas, and GCS activities improved by 60% to 70% in both areas. Thus, potentially toxic levels of brain ceramide induced by MCAO were attenuated to intermediate levels in preconditioned animals; brain GCS activity was relatively preserved. In ischemic tolerance, GCS appears to modulate otherwise high levels of brain ceramide.

Preconditioning with sublethal ischemia results in natural tolerance to ischemic stress, where multiple mediators of ischemic damage are simultaneously counteracted. Tumor necrosis factor alpha (TNF-alpha) has been implicated in development of ischemic tolerance. Using cellular models of ischemic tolerance, we have demonstrated that an effector of TNF-alpha-induced preconditioning is ceramide, a sphingolipid messenger in TNF-alpha signaling. TNF-alpha/ceramide-induced preconditioning protected cultured neurons against ischemic death and cultured astrocytes against proinflammatory effects of TNF-alpha. TNF-alpha activates a transcription factor NF-kappaB that binds promoters of multiple genes, thus ensuring pleiotropic effects of TNF-alpha. We describe here a mechanism that allows selective suppression of TNF-alpha/NF-kappaB-induced harmful genes in preconditioned cells while preserving cytoprotective responses. We demonstrate that in astrocytes activation of an adhesion molecule ICAM-1 by TNF-alpha is regulated through association of the phosphorylated p65 subunit of NF-kappaB with an adapter protein, p300, and that in preconditioned cells p65 remains unphosphorylated and ICAM-1 transcription is inhibited. However, TNF-alpha-activated transcription of a protective enzyme, MnSOD, does not depend on p300 and does not become inhibited in preconditioned cells. This new understanding of TNF-alpha-induced adaptation to ischemic stress and inflammation could suggest novel avenues for clinical intervention during ischemic and inflammatory diseases.