Postischemic progression of brain damage is multifactorial. We are analyzing a state in which nature has solved this complex problem. 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 cell loss. Efforts to isolate and identify the mechanisms that regulate the controlled metabolic depression and tolerance of profound brain ischemia that forms the essence of natural hibernation are in progress. 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 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 contribute 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. The small ubiquitin-like modifier (SUMO) leads to widespread effects in cell biology by post-translational modification of many proteins. We have found massive SUMOylation of proteins in body tissues during hibernation. 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 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 cells to oxygen/glucose deprivation (OGD), while the overexpression of a dominant negative mutant of Ubc9 sensitized the cells to OGD. These results suggested an important role of Ubc9 in cytoprotection against ischemia, probably through controlling SUMOylation levels. Conditional transgenic mice that express ubiquitin conjugating enzyme-9 (Ubc9), the E2 specific conjugase for SUMOylatiion of substrate protein, are being expanded. We will directly test the effects of SUMOylation levels on resistance to brain ischemia in these animals. We are also examining the effects of identified SUMOylated proteins on ischemic tolerance iin our established cell line models.
