Temporal Characteristics of Cytosolic Translocation of Mitochondrial Proteins in Permanent Distal Middle Cerebral Artery Occlusion Model of Rats

  • Shin, Byoung-Wook (Department of Neurosurgery, St. Vincent's Hospital, The Catholic University of Korea) ;
  • Sung, Jae-Hoon (Department of Neurosurgery, St. Vincent's Hospital, The Catholic University of Korea) ;
  • Hong, Jae-Taek (Department of Neurosurgery, St. Vincent's Hospital, The Catholic University of Korea) ;
  • Son, Byung-Chul (Department of Neurosurgery, St. Vincent's Hospital, The Catholic University of Korea) ;
  • Lee, Sang-Won (Department of Neurosurgery, St. Vincent's Hospital, The Catholic University of Korea) ;
  • Park, Chun-Kun (Department of Neurosurgery, St. Vincent's Hospital, The Catholic University of Korea)
  • Published : 2007.05.30

Abstract

Objective : In permanent distal middle cerebral artery occlusion [pdMCAO] model of rats, the temporal order of subcellular translocation is not fully understood yet. We studied translocation sequence of cytochrome c and apoptosis inducing factor [AIF] after pdMCAO and patterns of expression. Methods : Twenty-one male rats - with ten minutes, 1, 4, 8, 24 and 48 hours of pdMCAO groups - were enrolled. At core and penumbra area of each cerebral cortex, Western blotting of cytochrome c and AIF were performed using cytosolic fractions and then compared with sham specimens. With 48 hours group, the expression of cytochrome c and AIF was examined with immunofluorescent staining. Results : Compared to sham, the cytosolic translocation of cytochrome c significantly increased at all time points [p<0.05]. As early as 10 min after onset of ischemia, it was increased significantly [p<0.01]. The cytosolic translocation of AIF showed gradual increase with the passage of time and significantly increased 8 hours after [p<0.05]. As late as 24 hours and 48 hours after onset of ischemia, there were increased most significantly [p<0.01]. At penumbra, both proteins failed to show significant increase at all time points. At 48 hours after ischemia, colocalization of cytochrome c and AIF were confirmed. Conclusion : Cytosolic translocation of cytochrome c peaks much earlier than that of AIF in pdMCAO model of rat. Caspase dependent apoptosis activates soon after ischemia and later, it can be reinforced by gradually increasing AIF in ischemic core.

Keywords

References

  1. Arnoult D, Gaume B, Karbowski M, Sharpe JC, Cecconi F, Youle RJ : Mitochondrial release of AIF and EndoG requires caspase activation downstream of Bax/Bak-mediated permeabilization. Embo J 22 : 4385-4399, 2003 https://doi.org/10.1093/emboj/cdg423
  2. Arnoult D, Parone P, Martinou JC, Antonsson B, Estaquier J, Ameisen JC : Mitochondrial release of apoptosis-inducing factor occurs downstream of cytochrome c release in response to several proapoptotic stimuli. J Cell Biol 159 : 923-929, 2002 https://doi.org/10.1083/jcb.200207071
  3. Blomgren K, Zhu C, Hallin U, Hagberg H : Mitochondria and ischemic reperfusion damage in the adult and in the developing brain. Biochem Biophys Res Commun 304 : 551-559, 2003 https://doi.org/10.1016/S0006-291X(03)00628-4
  4. Bright R, Raval AP, Dembner JM, Perez-Pinzon MA, Steinberg GK, Yenari MA, et al : Protein kinase C delta mediates cerebral reperfusion injury in vivo. J Neurosci 24 : 6880-6888, 2004 https://doi.org/10.1523/JNEUROSCI.4474-03.2004
  5. Cao G, Clark RS, Pei W, Yin W, Zhang F, Sun FY, et al : Translocation of apoptosis-inducing factor in vulnerable neurons after transient cerebral ischemia and in neuronal cultures after oxygen-glucose deprivation. J Cereb Blood Flow Metab 23 : 1137-1150, 2003 https://doi.org/10.1097/01.WCB.0000087090.01171.E7
  6. Chen Q, Gong B, Almasan A : Distinct stages of cytochrome c release from mitochondria : evidence for a feedback amplification loop linking caspase activation to mitochondrial dysfunction in genotoxic stress induced apoptosis. Cell Death Differ 7 : 227-233, 2000 https://doi.org/10.1038/sj.cdd.4400629
  7. Cregan SP, Fortin A, MacLaurin JG, Callaghan SM, Cecconi F, Yu SW, et al : Apoptosis-inducing factor is involved in the regulation of caspaseindependent neuronal cell death. J Cell Biol 158 : 507-517, 2002 https://doi.org/10.1083/jcb.200202130
  8. Daugas E, Susin SA, Zamzami N, Ferri KF, Irinopoulou T, Larochette N, et al : Mitochondrio-nuclear translocation of AIF in apoptosis and necrosis. Faseb J 14 : 729-739, 2000 https://doi.org/10.1096/fasebj.14.5.729
  9. Fiskum G : Mitochondrial participation in ischemic and traumatic neural cell death. J Neurotrauma 17 : 843-855, 2000 https://doi.org/10.1089/neu.2000.17.843
  10. Fujimura M, Morita-Fujimura Y, Murakami K, Kawase M, Chan PH : Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats. J Cereb Blood Flow Metab 18 : 1239-1247, 1998 https://doi.org/10.1097/00004647-199811000-00010
  11. Fukuyama N, Takizawa S, Ishida H, Hoshiai K, Shinohara Y, Nakazawa H : Peroxynitrite formation in focal cerebral ischemia-reperfusion in rats occurs predominantly in the peri-infarct region. J Cereb Blood Flow Metab 18 : 123-129, 1998 https://doi.org/10.1097/00004647-199802000-00001
  12. Gill R, Soriano M, Blomgren K, Hagberg H, Wybrecht R, Miss MT, et al : Role of caspase-3 activation in cerebral ischemia-induced neurodegeneration in adult and neonatal brain. J Cereb Blood Flow Metab 22 : 420-430, 2002 https://doi.org/10.1097/00004647-200204000-00006
  13. Graham SH, Chen J : Programmed cell death in cerebral ischemia. J Cereb Blood Flow Metab 21 : 99-109, 2001 https://doi.org/10.1097/00004647-200102000-00001
  14. Green DR, Kroemer G : The pathophysiology of mitochondrial cell death. Science 305 : 626-629, 2004 https://doi.org/10.1126/science.1099320
  15. Hu BR, Liu CL, Ouyang Y, Blomgren K, Siesjo BK : Involvement of caspase-3 in cell death after hypoxia-ischemia declines during brain maturation. J Cereb Blood Flow Metab 20 : 1294-1300, 2000 https://doi.org/10.1097/00004647-200009000-00003
  16. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD : The release of cytochrome c from mitochondria : a primary site for Bcl-2 regulation of apoptosis. Science 275 : 1132-1136, 1997 https://doi.org/10.1126/science.275.5303.1132
  17. Li P, Nijhawan D, Budihardjo I, Srinivasula SM, Ahmad M, Alnemri ES, et al : Cytochrome c and dATP-dependent formation of Apaf- 1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91 : 479-489, 1997 https://doi.org/10.1016/S0092-8674(00)80434-1
  18. Li Y, Chopp M, Jiang N, Yao F, Zaloga C : Temporal profile of in situ DNA fragmentation after transient middle cerebral artery occlusion in the rat. J Cereb Blood Flow Metab 15 : 389-397, 1995 https://doi.org/10.1038/jcbfm.1995.49
  19. Lipton SA, Bossy-Wetzel E : Dueling activities of AIF in cell death versus survival : DNA binding and redox activity. Cell 111 : 147-150, 2002 https://doi.org/10.1016/S0092-8674(02)01046-2
  20. Liu X, Kim CN, Yang J, Jemmerson R, Wang X : Induction of apoptotic program in cell-free extracts : requirement for dATP and cytochrome c. Cell 86 : 147-157, 1996 https://doi.org/10.1016/S0092-8674(00)80085-9
  21. Loeffler M, Daugas E, Susin SA, Zamzami N, Metivier D, Nieminen AL, et al : Dominant cell death induction by extramitochondrially targeted apoptosis-inducing factor. Faseb J 15 : 758-767, 2001 https://doi.org/10.1096/fj.00-0388com
  22. MacManus JP, Linnik MD : Gene expression induced by cerebral ischemia : an apoptotic perspective. J Cereb Blood Flow Metab 17 : 815-832, 1997 https://doi.org/10.1038/aj.jcbfm.9590266
  23. Marzo I, Susin SA, Petit PX, Ravagnan L, Brenner C, Larochette N, et al : Caspases disrupt mitochondrial membrane barrier function. FEBS Lett 427 : 198-202, 1998 https://doi.org/10.1016/S0014-5793(98)00424-4
  24. Mayer A, Neupert W, Lill R : Translocation of apocytochrome c across the outer membrane of mitochondria. J Biol Chem 270 : 12390-12397, 1995 https://doi.org/10.1074/jbc.270.21.12390
  25. Newmeyer DD, Ferguson-Miller S : Mitochondria : releasing power for life and unleashing the machineries of death. Cell 112 : 481-490, 2003 https://doi.org/10.1016/S0092-8674(03)00116-8
  26. Ouyang YB, Tan Y, Comb M, Liu CL, Martone ME, Siesjo BK, et al : Survival- and death-promoting events after transient cerebral ischemia : phosphorylation of Akt, release of cytochrome C and Activation of caspaselike proteases. J Cereb Blood Flow Metab 19 : 1126-1135, 1999 https://doi.org/10.1097/00004647-199910000-00009
  27. Penninger JM, Kroemer G : Mitochondria, AIF and caspases-rivaling for cell death execution. Nat Cell Biol 5 : 97-99, 2003 https://doi.org/10.1038/ncb0203-97
  28. Scaffidi C, Fulda S, Srinivasan A, Friesen C, Li F, Tomaselli KJ, et al : Two CD95 (APO-1/Fas) signaling pathways. Embo J 17 : 1675- 1687, 1998 https://doi.org/10.1093/emboj/17.6.1675
  29. Shin CS, Choi BY, Jung ES, Kim SW, Chang CH, Cho SH : The time evolution of cerebral apoptosis in the permanent middle cerebral artery occlusion model of rats. J Korean Neurosurg Soc 37 : 54-58, 2005
  30. Sugawara T, Fujimura M, Morita-Fujimura Y, Kawase M, Chan PH : Mitochondrial release of cytochrome c corresponds to the selective vulnerability of hippocampal CA1 neurons in rats after transient global cerebral ischemia. J Neurosci 19 : RC39, 1999
  31. Susin SA, Daugas E, Ravagnan L, Samejima K, Zamzami N, Loeffler M, et al : Two distinct pathways leading to nuclear apoptosis. J Exp Med 192 : 571-580, 2000 https://doi.org/10.1084/jem.192.4.571
  32. Susin SA, Lorenzo HK, Zamzami N, Marzo I, Snow BE, Brothers GM, et al : Molecular characterization of mitochondrial apoptosisinducing factor. Nature 397 : 441-446, 1999 https://doi.org/10.1038/17135
  33. Xia T, Jiang C, Li L, Wu C, Chen Q, Liu SS : A study on permeability transition pore opening and cytochrome c release from mitochondria, induced by caspase-3 in vitro. FEBS Lett 510 : 62-66, 2002 https://doi.org/10.1016/S0014-5793(01)03228-8
  34. Yenari MA, Iwayama S, Cheng D, Sun GH, Fujimura M, Morita- Fujimura Y, et al : Mild hypothermia attenuates cytochrome c release but does not alter Bcl-2 expression or caspase activation after experimental stroke. J Cereb Blood Flow Metab 22 : 29-38, 2002 https://doi.org/10.1097/00004647-200201000-00004
  35. Yenari MA, Minami M, Sun GH, Meier TJ, Kunis DM, McLaughlin JR, et al : Calbindin d28k overexpression protects striatal neurons from transient focal cerebral ischemia. Stroke 32 : 1028-1035, 2001 https://doi.org/10.1161/01.STR.32.4.1028
  36. Yuan J, Horvitz HR : A first insight into the molecular mechanisms of apoptosis. Cell 116 : S53-56, 51 p following S59, 2004 https://doi.org/10.1016/S0092-8674(04)00028-5
  37. Zhao H, Shimohata T, Wang JQ, Sun G, Schaal DW, Sapolsky RM, et al : Akt contributes to neuroprotection by hypothermia against cerebral ischemia in rats. J Neurosci 25 : 9794-9806, 2005 https://doi.org/10.1523/JNEUROSCI.3163-05.2005
  38. Zhao H, Yenari MA, Cheng D, Barreto-Chang OL, Sapolsky RM, Steinberg GK : Bcl-2 transfection via herpes simplex virus blocks apoptosis-inducing factor translocation after focal ischemia in the rat. J Cereb Blood Flow Metab 24 : 681-692, 2004 https://doi.org/10.1097/01.WCB.0000127161.89708.A5
  39. Zhao H, Yenari MA, Cheng D, Sapolsky RM, Steinberg GK : Biphasic cytochrome c release after transient global ischemia and its inhibition by hypothermia. J Cereb Blood Flow Metab 25 : 1119-1129, 2005 https://doi.org/10.1038/sj.jcbfm.9600111
  40. Zhu C, Qiu L, Wang X, Hallin U, Cande C, Kroemer G, et al : Involvement of apoptosis-inducing factor in neuronal death after hypoxiaischemia in the neonatal rat brain. J Neurochem 86 : 306-317, 2003 https://doi.org/10.1046/j.1471-4159.2003.01832.x
  41. Zou H, Li Y, Liu X, Wang X : An APAF-1.cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9. J Biol Chem 274 : 11549-11556, 1999 https://doi.org/10.1074/jbc.274.17.11549