Ischemic preconditioning is a phenomenon in which low-level stressful stimuli upregulate endogenous defensive programs, resulting in subsequent resistance to otherwise lethal injuries. We previously observed that signal transduction systems typically associated with neurodegeneration such as caspase activation are requisite events for the expression of tolerance and induction of HSP70. In this work, we sought to determine the extent and duration of oxidative and energetic dysfunction as well as the role of effector kinases on metabolic function in preconditioned cells. Using an in vitro neuronal culture model, we observed a robust increase in Raf and p66Shc activation within 1 h of preconditioning. Total ATP content decreased by 25% 3 h after preconditioning but returned to baseline by 24 h. Use of a free radical spin trap or p66 shc inhibitor increased ATP content whereas a Raf inhibitor had no effect. Phosphorylated p66 shc rapidly relocalized to the mitochondria and in the absence of activated p66 shc , autophagic processing increased. The constitutively expressed chaperone HSC70 relocalized to autophagosomes. Preconditioned cells experience significant total oxidative stress measured by F 2 -isoprostanes and neuronal stress evaluated by F 4 -neuroprostane measurement. Neuroprostane levels were enhanced in the presence of Shc inhibitors. Finally, we found that inhibiting either p66 shc or Raf blocked neuroprotection afforded by preconditioning as well as upregulation of HSP70, suggesting both kinases are critical for preconditioning but function in fundamentally different ways. This is the first work to demonstrate the essential role of p66 shc in mediating requisite mitochondrial and energetic compensation after preconditioning and suggests a mechanism by which protein and organelle damage mediated by ROS can increase HSP70.

Metabolic adaptation to stress is a crucial yet poorly understood phenomenon, particularly in the central nervous system (CNS). The ability to identify essential metabolic events which predict neuronal fate in response to injury is critical to developing predictive markers of outcome, for interpreting CNS spectroscopic imaging, and for providing a richer understanding of the relevance of clinical indices of stress which are routinely collected. In this work, real-time multianalyte microphysiometry was used to dynamically assess multiple markers of aerobic and anaerobic respiration through simultaneous electrochemical measurement of extracellular glucose, lactate, oxygen, and acid. Pure neuronal cultures and mixed cultures of neurons and glia were compared following a 90 min exposure to aglycemia. This stress was cytotoxic to neurons yet resulted in no appreciable increase in cell death in age-matched mixed cultures. The metabolic profile of the cultures was similar in that aglycemia resulted in decreases in extracellular acidification and lactate release in both pure neurons and mixed cultures. However, oxygen consumption was only diminished in the neuron enriched cultures. The differences became more pronounced when cells were returned to glucose-containing media upon which extracellular acidification and oxygen consumption never returned to baseline in cells fated to die. Taken together, these data suggest that lactate release is not predictive of neuronal survival. Moreover, they reveal a previously unappreciated relationship of astrocytes in maintaining oxygen uptake and a correlation between metabolic recovery of neurons and extracellular acidification.

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