title: Essential role of the redox-sensitive kinase p66shc in determining energetic and oxidative status and cell fate in neuronal preconditioning.
abstract: 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.
authors.affiliation: Department of Neurology, Medicine and Vanderbilt Kennedy Center, Vanderbilt University, Nashville, Tennessee 37232, USA.
authors.forename: Jacquelynn E
authors.initials: JE
authors.lastname: Brown
authors.forename: Stephanie L H
authors.initials: SL
authors.lastname: Zeiger
authors.forename: Jane C
authors.initials: JC
authors.lastname: Hettinger
authors.forename: Joshua D
authors.initials: JD
authors.lastname: Brooks
authors.forename: Benjamin
authors.initials: B
authors.lastname: Holt
authors.forename: Jason D
authors.initials: JD
authors.lastname: Morrow
authors.forename: Erik S
authors.initials: ES
authors.lastname: Musiek
authors.forename: Ginger
authors.initials: G
authors.lastname: Milne
authors.forename: Bethann
authors.initials: B
authors.lastname: McLaughlin
mesh_terms: 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
chemical_list: 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
references.citation: Mol Biol Cell. 2004 Nov;15(11):4829-40
references.pmid: 15331765
references.citation: NeuroRx. 2004 Jan;1(1):26-35
references.pmid: 15717005
references.citation: J Neurochem. 2004 Apr;89(1):157-67
references.pmid: 15030400
references.citation: J Biol Chem. 2004 Jun 11;279(24):25689-95
references.pmid: 15078873
references.citation: Neurobiol Dis. 2008 Dec;32(3):329-39
references.pmid: 18760364
references.citation: Annu Rev Physiol. 2000;62:79-109
references.pmid: 10845085
references.citation: Prog Lipid Res. 1985;24(2):69-176
references.pmid: 3916238
references.citation: J Neurosci. 2008 Apr 16;28(16):4172-82
references.pmid: 18417696
references.citation: Proc Natl Acad Sci U S A. 2003 Jan 21;100(2):715-20
references.pmid: 12522260
references.citation: Cell. 1997 Apr 4;89(1):73-82
references.pmid: 9094716
references.citation: Neuropharmacology. 2008 Sep;55(3):334-44
references.pmid: 18402985
references.citation: Circulation. 1986 Nov;74(5):1124-36
references.pmid: 3769170
references.citation: J Neurochem. 1997 May;68(5):1836-45
references.pmid: 9109508
references.citation: Mol Cell Biol. 2008 Jul;28(13):4424-33
references.pmid: 18426906
references.citation: Cell Death Differ. 2007 Feb;14(2):338-47
references.pmid: 16794602
references.citation: Brain Res. 2000 Apr 28;863(1-2):169-81
references.pmid: 10773205
references.citation: J Cereb Blood Flow Metab. 1999 Nov;19(11):1229-37
references.pmid: 10566969
references.citation: Diabetes. 2005 Oct;54(10):2882-90
references.pmid: 16186389
references.citation: Autophagy. 2008 Apr;4(3):364-7
references.pmid: 18216498
references.citation: Proc Natl Acad Sci U S A. 2000 Jan 4;97(1):436-41
references.pmid: 10618436
references.citation: Int J Biochem Cell Biol. 2004 Dec;36(12):2435-44
references.pmid: 15325583
references.citation: Circulation. 2004 Jun 15;109(23):2917-23
references.pmid: 15173034
references.citation: Circ Res. 2004 Mar 5;94(4):420-32
references.pmid: 15001541
references.citation: Apoptosis. 2004 Mar;9(2):111-21
references.pmid: 15004508
references.citation: J Biol Chem. 2006 Jun 23;281(25):17220-17227
references.pmid: 16624816
references.citation: Free Radic Biol Med. 2005 Mar 15;38(6):711-8
references.pmid: 15721981
references.citation: Science. 2007 Feb 2;315(5812):659-63
references.pmid: 17272725
references.citation: Circ Res. 2002 Jul 12;91(1):38-45
references.pmid: 12114320
references.citation: Curr Biol. 2001 Mar 6;11(5):361-5
references.pmid: 11267874
references.citation: J Biol Chem. 2008 Apr 18;283(16):10892-903
references.pmid: 18281291
references.citation: Cell Mol Life Sci. 2002 May;59(5):808-20
references.pmid: 12088281
references.citation: Proc Natl Acad Sci U S A. 2007 Mar 20;104(12):5217-22
references.pmid: 17360381
references.citation: Chem Phys Lipids. 2004 Mar;128(1-2):117-24
references.pmid: 15037157
references.citation: J Cereb Blood Flow Metab. 1997 Feb;17(2):175-82
references.pmid: 9040497
references.citation: J Biol Chem. 2006 Apr 14;281(15):10555-60
references.pmid: 16481327
references.citation: J Neurochem. 2007 Nov;103(4):1472-80
references.pmid: 17680990
references.citation: Neuroreport. 2000 Apr 27;11(6):1357-60
references.pmid: 10817621
references.citation: EMBO J. 1997 Feb 17;16(4):706-16
references.pmid: 9049300
references.citation: J Biol Chem. 2000 Jun 16;275(24):18210-8
references.pmid: 10747973
references.citation: Brain Res Bull. 2000 Sep 15;53(2):141-6
references.pmid: 11044589
references.citation: Nat Cell Biol. 2001 Mar;3(3):276-82
references.pmid: 11231577
references.citation: Circulation. 1998 Jun 23;97(24):2463-9
references.pmid: 9641699
references.citation: J Biol Chem. 2000 Apr 21;275(16):12200-6
references.pmid: 10766856
references.citation: Brain Res. 1997 Feb 7;747(2):297-303
references.pmid: 9046005
references.citation: Arterioscler Thromb Vasc Biol. 2008 Apr;28(4):622-8
references.pmid: 18162611
references.citation: Autophagy. 2008 Feb;4(2):151-75
references.pmid: 18188003
references.citation: J Neurochem. 2008 May;105(4):1525-35
references.pmid: 18248613
references.citation: Physiol Rev. 2008 Jan;88(1):211-47
references.pmid: 18195087
references.citation: J Cereb Blood Flow Metab. 2003 Sep;23(9):1052-9
references.pmid: 12973021
references.citation: J Hepatol. 2003 Dec;39(6):972-7
references.pmid: 14642614
references.citation: FASEB J. 2001 Oct;15(12):2286-7
references.pmid: 11511528
references.citation: Proc Natl Acad Sci U S A. 2005 Sep 27;102(39):13807-12
references.pmid: 16174725
references.citation: J Cereb Blood Flow Metab. 2004 Jun;24(6):636-45
references.pmid: 15181371
references.citation: Biol Chem. 2006 Oct-Nov;387(10-11):1405-10
references.pmid: 17081113
references.citation: J Biol Chem. 2005 Apr 1;280(13):13148-52
references.pmid: 15671027
references.citation: J Neurosci. 2007 Aug 1;27(31):8238-49
references.pmid: 17670970
references.citation: Mol Cell Biol. 1998 Nov;18(11):6624-33
references.pmid: 9774677
references.citation: Proc Natl Acad Sci U S A. 2001 Jul 3;98(14):7783-8
references.pmid: 11427728
references.citation: Nature. 1999 Nov 18;402(6759):309-13
references.pmid: 10580504
references.citation: Transplant Proc. 2003 Aug;35(5):1800-2
references.pmid: 12962800
references.citation: Nat Rev Mol Cell Biol. 2005 Jun;6(6):505-10
references.pmid: 15928714
references.citation: Free Radic Biol Med. 2005 Mar 15;38(6):698-710
references.pmid: 15721980
references.citation: Circulation. 2005 Sep 27;112(13):1971-8
references.pmid: 16172266
references.citation: Cell. 2005 Jul 29;122(2):221-33
references.pmid: 16051147
references.citation: J Neurochem. 2006 Jun;97(5):1301-13
references.pmid: 16638022
references.citation: J Cereb Blood Flow Metab. 1996 Jul;16(4):566-77
references.pmid: 8964795
references.citation: Free Radic Biol Med. 2009 Nov 15;47(10):1422-31
references.pmid: 19699297
references.citation: Am J Physiol Heart Circ Physiol. 2007 Jun;292(6):H2573-81
references.pmid: 17337592
references.citation: J Neurosci Methods. 2001 Jan 15;104(2):165-76
references.pmid: 11164242
references.citation: J Neurochem. 2000 Dec;75(6):2401-8
references.pmid: 11080191
references.citation: Biochem Biophys Res Commun. 2009 Feb 6;379(2):293-7
references.pmid: 19103170
references.citation: EMBO J. 1999 Apr 15;18(8):2137-48
references.pmid: 10205168
references.citation: Stroke. 2003 Jul;34(7):1796-802
references.pmid: 12791941
references.citation: EMBO J. 2000 Nov 1;19(21):5720-8
references.pmid: 11060023
references.citation: J Neurosci. 2002 Aug 1;22(15):6401-7
references.pmid: 12151519
references.citation: Endocr Relat Cancer. 2006 Dec;13 Suppl 1:S125-35
references.pmid: 17259553
references.citation: Ann Neurol. 2000 Sep;48(3):285-96
references.pmid: 10976634
references.citation: Brain Res. 1990 Sep 24;528(1):21-4
references.pmid: 2245337
references.citation: J Biol Chem. 2000 Feb 4;275(5):3722-8
references.pmid: 10652372
references.citation: Proc Natl Acad Sci U S A. 1990 Dec;87(23):9383-7
references.pmid: 2123555
references.citation: Nat Protoc. 2007;2(1):221-6
references.pmid: 17401357