Abstract
Morphological evidence of apoptosis in transient forebrain ischemia is controversial. We therefore investigated the time sequence of apoptosis‐related antigens by immunohistochemistry and correlated it with emerging nuclear patterns of cell death in a model of transient forebrain ischemia in CA1 pyramidal cells of the rat hippocampus. The earliest ischemic changes were found on day 2 and 3, reflected by an upregulation of phospho‐c‐Jun in a proportion of morphologically intact CA1 neurons, which matched the number of neurons that succumbed to ischemia at later time points. At day 3 and later 3 ischemic cell death morphologies became apparent: pyknosis, apoptosis‐like cell death and necrosis‐like cell death, which were confirmed by electron microscopy. Activated caspase‐3 was present in the vast majority of cells with apoptosis‐like morphology as well as in a small subset of cells undergoing necrosis; its expression peaked on days 3 to 4. Silver staining for nucleoli, which are a substrate for caspase‐3, revealed a profound loss of nucleoli in cells with apoptosis‐like morphology, whereas cells with necrosis‐like morphology showed intact nucleoli. Overall, cells with apoptosis‐like morphology and/or caspase‐3 expression represented a minor fraction (<10%) of ischemic neurons, while the vast majority followed a necrosis‐like pathway. Our studies suggest that CA1 pyramidal cell death following transient forebrain ischemia may be initiated through c‐Jun N‐terminal kinase (JNK) pathway activation, which then either follows an apoptosis‐like cell death pathway or leads to secondary necrosis.
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REFERENCES
- 1. Behrens A, Sibilia M, Wagner EF (1999) Amino‐terminal phosphorylation of c‐Jun regulates stress‐induced apoptosis and cellular proliferation. Nat Genet 21:326–329. [DOI] [PubMed] [Google Scholar]
- 2. Bering R, Diemer NH, Draguhn A, Johansen FF (1995) Co‐localization of somatostatin mRNA and parvalbumin in dorsal rat hippocampus after cerebral ischemia. Hippocampus 5:341–348. [DOI] [PubMed] [Google Scholar]
- 3. Biggiogera M, Bottone MG, Pellicciari C (1997) Nuclear ribonucleoprotein‐containing structures undergo severe rearrangement during spontaneous thymocyte apoptosis. A morphological study by electron microscopy. Histochem Cell Biol 107:331–336. [DOI] [PubMed] [Google Scholar]
- 4. Borsello T, Clarke PG, Hirt L, Vercelli A, Repici M, Schorderet DF, Bogousslavsky J, Bonny C (2003) A peptide inhibitor of c‐Jun N‐terminal kinase protects against excitotoxicity and cerebral ischemia. Nat Med 9:1180–1186. [DOI] [PubMed] [Google Scholar]
- 5. Cao G, Luo Y, Nagayama T, Pei W, Stetler RA, Graham SH, Chen J (2002) Cloning and characterization of rat caspase‐9: implications for a role in mediating caspase‐3 activation and hippocampal cell death after transient cerebral ischemia. J Cereb Blood Flow Metab 22:534–546. [DOI] [PubMed] [Google Scholar]
- 6. Carboni S, Hiver A, Szyndralewiez C, Gaillard P, Gotteland JP, Vitte PA (2004) AS601245 (1,3‐ben‐zothiazol‐2‐yl (2‐[[2‐(3‐pyridinyl) ethyl] amino]‐4 pyrimidinyl) acetonitrile): a c‐Jun NH2‐terminal protein kinase inhibitor with neuroprotective properties. J Pharmacol Exp Ther 310:25–32. [DOI] [PubMed] [Google Scholar]
- 7. Chen J, Nagayama T, Jin K, Stetler RA, Zhu RL, Graham SH, Simon RP (1998). Induction of cas‐pase‐3‐like protease may mediate delayed neuronal death in the hippocampus after transient cerebral ischemia. J Neurosci 18:4914–4928. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8. Colbourne F, Sutherland GR, Auer RN (1999) Electron microscopic evidence aganist apoptosis as the mechanism of neuronal death in global ischemia. J Neurosci 19:4200–4210. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Columbaro M, Gobbi P, Renò F, Luchetti F, Santi S, Valmori A, Falcieri E (1999) A multiple technical approach to the study of apoptotic cell micronuclei. Scanning 8:541–548. [DOI] [PubMed] [Google Scholar]
- 10. Desphande JK, Bergstedt K, Lindén T, Kalimo H, Wieloch T (1992) Ultrastructural changes in the hippocampal CA1 region following transient cerebral ischemia: evidence aganinst programmed cell death. Exp Brain Res 88:91–105. [DOI] [PubMed] [Google Scholar]
- 11. Endres M, Namura S, Shimizu‐Sasamata M, Waeber C, Zhang L, Gómez‐Isla T, Hyman BT, Moskowitz MA (1998) Attenuation of delayed neuronal death after mild focal ischemia in mice by inhibition of the caspase family. J Cereb Blood Flow Metab 18:238–247. [DOI] [PubMed] [Google Scholar]
- 12. Estus S, Zaks WJ, Freeman RS, Gruda M, Bravo R, Johnson EM Jr (1994) Altered gene expression in neurons during programmed cell death: identification of c‐jun as necessary for neuronal apoptosis. J Cell Biol 127:1717–1727. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13. Evert BO, Wullner U, Klockgether T (2000) Cell death in polyglutamine diseases. Cell Tissue Res 301:189–204. [DOI] [PubMed] [Google Scholar]
- 14. Ferrer I, Pozas E, Marti M, Blanco R, Planas AM (1997) Methyl‐azoxymethanol acetate‐induced apoptosis in the external granule layer of the developing cerebellum of the rat is associated with strong c‐Jun expression and formation of high molecular weight c‐Jun complexes. J Neuropathol Exp Neurol 56:1–9. [DOI] [PubMed] [Google Scholar]
- 15. Foghsgaard L, Wissing D, Mauch D, Lademann U, Bastholm L, Boes M, Elling F, Leist M, Jäättelä M (2001) Cathepsin B acts as a dominant execution protease in tumor cell apoptosis induced by tumor necrosis factor. J Cell Biol 153:999–1010. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16. Garrah JM, Bisby MA, Rossiter JP (1998) Immunolabelling of the cytoplasm and processes of apoptotic facial motorneurons following axotomy in the neonatal rat. Acta Neuropathol 95:223–228. [DOI] [PubMed] [Google Scholar]
- 17. Gillardon F, Kiprianova I, Sandkühler J, Hoss‐mann K‐A, Spranger M (1999) Inhibition of cas‐pases prevents cell death of hippocampal CA 1 neurons, but not impairment of hippocampal long‐term potentiation following global ischemia. Neuroscience 93:1219–1222. [DOI] [PubMed] [Google Scholar]
- 18. Gillardon F, Böttiger B, Schmitz B, Zimmer‐mann M, Hossmann KA (1997) Activation of CPP‐32 protease in hippocampal neurons following ischemia and epilepsy. Mol Brain Res 50:16–22. [DOI] [PubMed] [Google Scholar]
- 19. Gillardon F, Spranger M, Tiesler C, Hossmann KA (1999). Expression of cell death‐associated phospho‐c‐Jun and p53‐activated gene 608 in hippocampal CA 1 neurons following global ischemia. Mol Brain Res 73:138–143. [DOI] [PubMed] [Google Scholar]
- 20. Gold R, Schmid M, Giegerich G, Breitschopf H, Hartung HP, Toyka KV, Lassmann H (1994) Differentiation between cellular apoptosis and necrosis by combined use of in situ tailing and nick translation techniques. Lab Invest 71:219–225. [PubMed] [Google Scholar]
- 21. Gorman AM, Ceccatelli S, Orrenius S (2000) Role of mitochondria in neuronal apoptosis. Dev Neurosci 22:348–358. [DOI] [PubMed] [Google Scholar]
- 22. Ham J, Eilers A, Whitfield J, Neame SJ, Shah B (2000) c‐Jun and the transcriptional control of neuronal apoptosis. Biochem Pharmacol 60, 1015–1021. [DOI] [PubMed] [Google Scholar]
- 23. Han BH, Xu D, Choi J, Han Y, Xanthoudakis S, Roy S, Tam J, Vaillancourt J, Colucci J, Siman R, Giroux A, Robertson GS, Zamboni R, Nicholson DW, Holtzman DM (2002) Selective, reversible caspase‐3 inhibitor is neuroprotective and reveals distinct pathways of cell death after neonatal hypoxic‐ischemic brain injury. J Biol Chem 277:30128–30136. [DOI] [PubMed] [Google Scholar]
- 24. Himi T, Ishizaki Y, Murota S (1998) A caspase inhibitor blocks ischaemia‐induced delayed neuronal death in the gerbil. Eur J Neurosci 10:777–781. [DOI] [PubMed] [Google Scholar]
- 25. Horky M, Wurzer G, Kotala V, Anton M, Vojtesek B, Vacha J, Wesierska‐Gadek J (2001) Segregation of nucleolar components coincides with caspase‐3 activation in cisplatin‐treated HeLa cells. J Cell Sci 114:663–670. [DOI] [PubMed] [Google Scholar]
- 26. Horn M, Schlote W (1992) Delayed neuronal death and delayed neuronal recovery in the human brain following global ischemia. Acta Neuropathol 85:79–87. [DOI] [PubMed] [Google Scholar]
- 27. Ito U, Spat M, Walker JT Jr, Klatzo I (1975) Experimental cerebral ischemia in Mongolian gerbils: I. Light microscopic observations. Acta Neuropathol 32:209–233. [DOI] [PubMed] [Google Scholar]
- 28. Jäättelä M, Tschopp J (2003) Caspase‐independent cell death in T lymphocytes. Nat Immunol 4:416–423. [DOI] [PubMed] [Google Scholar]
- 29. Johansen FF (1993) Interneurons in rat hippocampus after cerebral ischemia. Morphometric, functional, and therapeutic investigations. Acta Neurol Scand Suppl 150:1–32. [PubMed] [Google Scholar]
- 30. Kallunki T, Su B, Tsigelny I, Sluss HK, Derijard B, Moore G, Davis R, Karin M (1994) JNK2 contains a specificity‐determining region responsible for efficient c‐Jun binding and phosphorylation. Genes Dev 8:2996–3007. [DOI] [PubMed] [Google Scholar]
- 31. Keane RW, Srinivasan A, Foster LM, Testa MP, Ord R, Nonner D, Wang HG, Reed JC, Bredesen DE, Kayalar C (1997) Activation of CPP32 during apoptosis of neurons and astrocytes. J Neurosci Res 48:168–180. [DOI] [PubMed] [Google Scholar]
- 32. Kerr JFR, Wyllie AH, Currie AR (1972) Apoptosis: a basic biological phenomenon with wide‐ranging implications in tissue kinetics. Br J Cancer 26:239–257. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 33. Kirino T (1982) Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res 239:57–69. [DOI] [PubMed] [Google Scholar]
- 34. Kirino T, Sano K (1984) Selective vulnerability in the gerbil hippocampus following transient ischemia. Acta Neuropathol 62:201–208. [DOI] [PubMed] [Google Scholar]
- 35. Kuan CY, Whitmarsh AJ, Yang DD, Liao G, Schloemer AJ, Dong C, Bao J, Banasiak KJ, Haddad GG, Flavell RA, Davis RJ, Rakic P (2003) A critical role of neural‐specific JNK3 for ischemic apoptosis. Proc Natl Acad Sci U S A 100:15184–15189. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 36. Leist M, Jäättelä M (2001) Four deaths and a funeral: from caspases to alternative mechanisms. Nat Rev Mol Cell Biol 2:589–598. [DOI] [PubMed] [Google Scholar]
- 37. Li H, Colbourne F, Sun P, Zhao Z, Buchan AM, Iadecola C (2000) Caspase inhibitors reduce neuronal injury after focal but not global ischemia in rats. Stroke 31:176–182. [DOI] [PubMed] [Google Scholar]
- 38. Lindner LE (1993) Improvements in the silver‐staining technique for nucleolar organizer regions (AgNOR). J Histochem Cytochem 41:439–445. [DOI] [PubMed] [Google Scholar]
- 39. Lockshin RA, Zakeri Z (2001) Programmed cell death and apoptosis: origins of the theory. Nat Rev Mol Cell Biol 2:545–550. [DOI] [PubMed] [Google Scholar]
- 40. Martelli AM, Robuffo I, Bortul R, Ochs RL, Luchetti F, Cocco L, Zweyer M, Bareggi R, Falcieri E (2000) Behavior of nucleolar proteins during the course of apoptosis in camptothecin‐treated HL60 cells. J Cell Biochem 78:264–277. [PubMed] [Google Scholar]
- 41. Mathiasen IS, Jäättelä M (2002) Triggering caspase‐independent cell death to combat cancer. Trends Mol Med 8:212–220. [DOI] [PubMed] [Google Scholar]
- 42. Matsuoka Y, Okazaki M, Zhao H, Asai S, Ishikawa K, Kitamura Y (1999) Phosphorylation of c‐Jun and its localization with heme oxygenase‐1 and cyclooxygenase‐2 in CA 1 pyramidal neurons after transient forebrain ischemia. J Cereb Blood Flow Metab 19:1247–1255. [DOI] [PubMed] [Google Scholar]
- 43. Minden A, Lin A, Smeal T, Derijard B, Cobb M, Davis R, Karin M. (1994) c‐Jun N‐terminal phosphorylation correlates with activation of the JNK subgroup but not the ERK subgroup of mitogen‐activated protein kinases. Mol Cell Biol 14:6683–6688. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 44. Ni B, Wu X, Su Y, Stephenson D, Smalstig EB, Clemens J, Paul SM (1998) Transient global fore‐brain ischemia induces a prolonged expression of the caspase‐3 mRNA in rat hippocampal CA 1 pyramidal neurons. J Cereb Blood Flow Metab 18:248–256. [DOI] [PubMed] [Google Scholar]
- 45. Nitatori T, Sato N, Waguri S, Karasawa Y, Araki H, Shibanai K, Kominami E, Uchiyama Y (1995) Delayed neuronal death in the CA1 pyramidal cell layer of the gerbil hippocampus following transient ischemia is apoptosis. J Neurosci 15:1001–1011. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 46. Petito CK, Feldmann E, Pulsinelli WA, Plum F (1987) Delayed hippocampal damage in humans following cardiorespiratory arrest. Neurology 37:1281–1286. [DOI] [PubMed] [Google Scholar]
- 47. Pulsinelli WA, Brierley JB, Plum F (1982) Temporal profile of neuronal damage in a model of transient forebrain ischemia. Ann Neurol 11:491–499. [DOI] [PubMed] [Google Scholar]
- 48. Rami A, Agarwal R, Botez G, Winckler J (2000) mu‐Calpain activation, DNA fragmentation, and synergistic effects of caspase and calpain inhibitors in protecting hippocampal neurons from ischemic damage. Brain Res 866:299–312. [DOI] [PubMed] [Google Scholar]
- 49. Schotte P, Declercq W, Van Huffel S, Vandenabeele P, Beyaert R (1999) Non‐specific effects of methyl ketone peptide inhibitors of caspases. FEBS Lett 442:117–121. [DOI] [PubMed] [Google Scholar]
- 50. Smith M‐L, Bendek G, Dahlgren N, Rosen I, Wieloch T, Siesjö BK (1984) Models for studying long‐term recovery following forebrain ischemia in the rat. A 2‐vessel occlusion model. Acta Neurol Scand 69:385–401. [DOI] [PubMed] [Google Scholar]
- 51. Srinivasan A, Roth KA, Sayers RO, Shindler KS, Wong AM, Fritz LC, Tomaselli K (1998) In situ immunodetection of activated caspase‐3 in apoptotic neurons in the developing nervous system. Cell Death Differ 5:1004–1016. [DOI] [PubMed] [Google Scholar]
- 52. Stadelmann C, Mews I, Srinivasan A, Deckwerth TL, Lassmann H, Brück W (2001) Expression of cell death‐associated proteins in neuronal apoptosis associated with pontosubicular neuron necrosis. Brain Pathol 11:273–281. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 53. Stadelmann C, Brück W, Bancher C, Jellinger K, Lassmann H (1998) Alzheimer disease: DNA fragmentation indicates increased neuronal vulnerability, but not apoptosis. J Neuropathol Exp Neurol 5:456–464. [DOI] [PubMed] [Google Scholar]
- 54. Stadelmann C, Deckwerth TL, Srinivasan A, Bancher C, Brück W, Jellinger K, Lassmann H (1999) Activation of caspase‐3 in single neurons and authophagic granules of granulovacuoler degeneration in Alzheimer's disease. Am J Pathol 155:1459–1466. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 55. Vancompernolle K, Van Herreweghe F, Pyn‐aert G, Van de Craen M, De Vos K, Totty N, Sterling A, Fiers W, Vandenabeele P, Grooten J (1998) Atractyloside‐induced release of cathepsin B, a protease with caspase‐processing activity. FEBS Lett 438:150–158. [DOI] [PubMed] [Google Scholar]
- 56. Wyllie AH, Kerr JFR, Currie AR (1980) Cell death: the significance of apoptosis. Int Rev Cytol 68:251–306. [DOI] [PubMed] [Google Scholar]
- 57. Yamashima T (2000) Implications of cysteine proteases calpain, cathepsin and scaspse in ischemic neuronal death of primates. Progress Neurobiol 62:273–295. [DOI] [PubMed] [Google Scholar]
- 58. Yuan J, Yankner BY (2000) Apoptosis in the nervous system. Nature 407:802–809. [DOI] [PubMed] [Google Scholar]
- 59. Zeng Y‐S, Xu Z‐C (2000) Co‐ecistence of necrosis and apoptosis in rat hippocampus following transient forebrain ischemia. Neurosci Res 37:113–125. [DOI] [PubMed] [Google Scholar]
- 60. Zhan R‐Z, Wu C, Fujihara H, Taga K, Qi S, Naito M, Shimoji K (2001) Both caspase dependent and caspase‐independent pathways may be involved in hippocampal CA 1 neuronal death because of loss of cytochrome c from mitochondria in a rat forebrain ischemia model. J Cereb Blood Flow Metabol 21:529–540. [DOI] [PubMed] [Google Scholar]