Essential role of the redox-sensitive kinase p66shc in determining energetic and oxidative status and cell fate in neuronal preconditioning.
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 p66(Shc) 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.
Brown
Jacquelynn E
JE
Department of Neurology, Medicine and Vanderbilt Kennedy Center, Vanderbilt University, Nashville, Tennessee 37232, USA.
Zeiger
Stephanie L H
SL
Hettinger
Jane C
JC
Brooks
Joshua D
JD
Holt
Benjamin
B
Morrow
Jason D
JD
Musiek
Erik S
ES
Milne
Ginger
G
McLaughlin
Bethann
B
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001343:Autophagy / Q000187:drug effects / Q000502:physiology; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D002478:Cells, Cultured; D004281:Docosahexaenoic Acids / Q000378:metabolism; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D019194:Ischemic Preconditioning; D028421:Isoprostanes / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D009474:Neurons / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D010766:Phosphorylation; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013997:Time Factors; D048490:raf Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
D050883:HSC70 Heat-Shock Proteins; C494373:Hspa8 protein, rat; D028421:Isoprostanes; D017382:Reactive Oxygen Species; D055765:Shc Signaling Adaptor Proteins; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D004281:Docosahexaenoic Acids; D000255:Adenosine Triphosphate; D048490:raf Kinases
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