Postischemic progression of brain damage is extremely multifactorial. We are analyzing a state in which nature has solved this problematic biocomplexity. Hibernation with its metabolic, hematologic and cell membrane adjustments permits animals to withstand extremely low blood flow in the brain for protracted periods with no cellular loss. Efforts to isolate and identify the mechanisms that regulate the controlled metabolic depression and tolerance of profound brain hypoperfusion that forms the essence of natural hibernation have been conducted. In hippocampal slices, hibernation confers robust resistance to hypoxia and glucose deprivation as compared to slices from non-hibernating ground squirrels and rats at 37 degrees C, 20 degrees C and 7 degrees C. These findings indicate that hibernation involves tolerance to an in vitro form of ischemic stress that is not strictly dependent on temperature. Protein synthesis (PS) in hippocampal slices is greatly depressed at the same incubation temperatures. PS in vivo was below the limit of autoradiographic detection in brain sections, and in brain extracts was determined to be 0.04% of the average rate from active squirrels. Further, it was threefold reduced in cell-free extracts from hibernating brain at 37 degrees C, eliminating hypothermia as the only cause for protein synthesis inhibition. PS suppression involved blocks of both initiation and elongation and its onset coincided with the entrance phase of the hibernation bout. An increased monosome peak with moderate ribosomal disaggregation in polysome profiles and the greatly increased phosphorylation of eIF2a are both consistent with an initiation block in hibernators. The elongation block was demonstrated by a threefold increase in ribosomal mean transit times in cell-free extracts from hibernators. Phosphorylation of eEF2 is increased, eEF2 kinase activity is increased, and protein phosphatase 2A activity is decreased during hibernation which contributes to the elongation block. No abnormalities of ribosomal function or mRNA levels were detected. These findings implicate suppression of PS as a component of the regulated shutdown of cellular function that permits hibernating ground squirrels to tolerate trickle blood flow and reduced substrate and oxygen availability. Further study of the factors that control these phenomena may lead to identification of the molecular mechanisms that regulate this state. Dephosphorylation of Akt/PKB has been found to occur in multiple tissues during hibernation. We find that depending on cellular contex in mammalian cells, cytoprotection can be associated with strong Akt activation, modulated Akt activation or modulated Akt inhibition. By cloning c-fos cDNA from the 13-lined ground squirrel (Spermophilus tridecemlineatus) and using squirrel c-fos mRNA probe for in situ hybridization histochemistry, we systematically analyzed and identified specific brain regions that were activated during six different phases of the hibernation bout. During entrance into torpor, we detected activation of the ventrolateral subdivision of the medial preoptic area ('thermoregulatory center'), and the reticular thalamic nucleus, which is known to inhibit the somatomotor cortex. During torpor, c-fos expression in the cortex was suppressed while the reticular thalamic nucleus remained uniformly active. Throughout torpor the suprachiasmatic nucleus ('biological clock') showed increasing activity, likely participating in phase-change regulation of the hibernation bout. Interestingly, during torpor very strong c-fos activation was seen in the epithelial cells of the choroid plexus and in tanycytes at the third ventricle, both peaking near the beginning of arousal. In arousal, activity of the suprachiasmatic and reticular thalamic nuclei and choroid epithelial cells diminished, while ependymal cells in the lateral and fourth ventricles showed stronger activity. Increasing body temperature during arousal was driven by the activation of neurons in the medial part of the preoptic area. In interbout awake animals, we demonstrated the activation of hypothalamic neurons located in the arcuate nucleus and the dorsolateral hypothalamus, areas involved in food intake. Our observations indicate that the hibernation bout is closely regulated and orchestrated by specific regions of the central nervous system. The small ubiquitin-like modifier (SUMO) leads to widespread effects in cell biology by post-translational modification of many proteins; it functions to preserve homeostasis under stress. We have found massive global SUMOylation of proteins in body tissues during hibernation torpor. We have also found that SUMOylation is essential for cell survival and that maintained or augmented SUMOylation is robustly cytoprotective in cell culture systems. The expression level of ubiquitin conjugating enzyme-9 (Ubc9) protein, the single SUMO-conjugating enzyme, was well correlated with the SUMOylation levels in the squirrels during hibernation bouts. In addition, the overexpression of Ubc9 by transfection enhanced the tolerance of SHSY5Y human neuroblastoma cells to oxygen/glucose deprivation (OGD), while the overexpression of a dominant negative mutant of Ubc9 sensitized the cells to OGD. Further experiments in primary neuronal cultures have shown that overexpression of SUMO1 and SUMO2 are cytoprotective and that knock down of SUMO1 with siRNA increases cell death during OGD. Transgenic mice that overexpress Ubc9, the E2 specific conjugase for SUMOylatiion of proteins, are showing that modest increases of Ubc9 increase global SUMOylation levels and confer a corresponding level of resistance to brain ischemia. We have established that the level of global SUMOylation is directly proportional to the level of cytoprotection in preclinical models of stroke. We have also examined the effects of SUMO-1 and SUMO-2/3 SUMOylated proteins on ischemic tolerance in our cell culture models. We have obtained clear data supporting a role for SUMOylation in ischemic tolerance. Current work adresses the study of molecular mechanisms that can control the level of global SUMOylation. We seek to boost global SUMOylation to levels seen in hibernating animals to harness the extraordinary cytoprotection inherent in this form of post-translational modification. To this end, we have identified microRNAs as regulators of both global SUMOylation and the post-translational modification levels with other ubiquitin-like modifiers. We have developed several high throughput assays for screening of compound libraries. We have identified a group of compounds with a robust capacity to globally SUMOylate proteins and are testing these compounds by means of in vitro and in vivo models of brain ischemia. We have identified molecular entities that can increase gobal SUMOylation and cytoprotect cultured neurons exposed to in vitro oxygen-glucose deprivation. We are also investigating cytoprotective changes in cell state related to such signaling.
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