[en] In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke.
Disciplines :
Neurology
Author, co-author :
Schulz, Jörg B.; Experimental Neuropharmacology Laboratory, Department of Neurology, University of Tübingen, Hoppe-Seyler-Str. 3, D-72076 Tübingen, Germany.jeorg.b.schulz@uni-tuebingen.de
Weller, Michael; Exp. Neuropharmacology Laboratory, Department of Neurology, University of Tübingen, D-72076 Tübingen, Germany
Matthews, Russell T.; Exp. Neuropharmacology Laboratory, Department of Neurology, University of Tübingen, D-72076 Tübingen, Germany ; Neurochemistry Laboratory, Massachusetts General Hospital, Harvard Medical School, Boston, MA 02114, United States
HENEKA, Michael ; Exp. Neuropharmacology Laboratory, Department of Neurology, University of Tübingen, D-72076 Tübingen, Germany
Groscurth, Peter; Institute of Anatomy, Division of Cell Biology, University of Zurich, Ch-8057 Zürich, Switzerland
This work was supported by grants from the Deutsche Forschungsge-meinschaft (Schu 932/2-1) and the fortüne program of the University Tübingen. We thank L. Dumitrescu and I. Müller for excellent technical assistance.
Alnemn E, Livingston D, Nicholson D, Salvesen G, Thornberry N, Wong W and Yuan J (1996) Human ICE/CED-3 protease nomenclature. Cell 87: 171
Ankarcrona M, Dypbukt JM, Bonfoco E, Zhivotovsky B, Orrenius S, Lipton SA and Nicotera P (1995) Glutamate-induced neuronal death: a succession of necrosis and apoptosis depending on mitochondrial function. Neuron 15: 961-973
Armstrong RC, Aja T, Xiang JL, Gaur S, Krebs JF, Hoang K, Bai X, Korsmeyer J, Karanewsky DS, Fritz LC and Tomaselli KJ (1996) Fas induced activation of the cell death related protease CPP32 is inhibited by Bcl-2 and by ICE family protease inhibitors. J. Biol. Chem. 271: 16850-16855
Armstrong RC, Aja TJ, Hoang KD, Gaur S, Bai X, Alnemn ES, Litwack G, Karanewsky DS, Fritz LC and Tomaselli KJ (1997) Activation of the CED3/ICE-related protease CPP32 in cerebellar granule neurons undergoing apoptosis but not necrosis. J. Neurosci. 17: 553-562
Beal MF, Brouillet E, Jenkins B, Henshaw R, Rosen B and Hyman BT (1993) Age-dependent stnatal excitotoxic lesions produced by the endogenous mitochondrial inhibitor malonate. J. Neurochem. 61: 1147-1150
Bhat RT, DiRocco R, Marcy VR, Flood DG, Zhu Y, Dobrzanski P, Siman R, Scott R, Contreras PC and Miller M (1996) Increased expression of IL-1β converting enzyme in hippocampus after ischemia: selective localization in microglia. J Neurosci. 16: 4146-4154
Bonfoco E, Krainc D, Ankarcrona M, Nicotera P and Lipton SA (1995) Apoptosis and necrosis: two distinct events induced, respectively, by mild and intense insults with N-methyl-D-aspartate or nitric oxide/superoxide in cortical cell cultures. Proc. Natl. Acad. Sci. USA 92: 7162-7166
Campagne M and Gill R (1996) Ultrastructural morphological changes are not characteristic of apoptotic cell death following focal cerebral ischaemia in the rat. Neurosci. Lett 213: 111-114
Cohen GM (1997) Caspases. the executioners of apoptosis Biochem. J 326: 1-16
Fernandes-Alnemri T, Armstrong RC, Krebs J, Srinivasula SM, Wang L, Bullrich F, Fritz LC, Trapani JA, Tomaselli KJ, Litwack G and Alnemri ES (1996) In vitro activation of CPP32 and Mch3 by Mch4, a novel human apoptotic cysteine protease containing two FADD-like domains. Proc. Natl. Acad Sci USA 93: 7464-7469
Friedlander RM, Gagliardini V, Hara H, Fink KB, Li W, MacDonald G, Fishman MC, Greenberg AH, Moskowitz MA and Yuan J (1997) Expression of a dominant negative mutant of interleukm-1β converting enzyme in transgenic mice prevents neuronal cell death induced by trophic factor withdrawal and ischemic brain injury. J. Exp. Med. 185: 933-940
Garcia JH, Liu K-F and Relton JK (1995) Interleukin-1 receptor antagonist decreases the number of necrotic neurons in rats with middle cerebral artery occlusion Am. J Pathol. 147: 1477-1486
Greco RW, Bravo G and Parsons JC (1995) The search for synergy a critical review from a response surface perspective. Pharmacol. Rev. 47: 331-385
Greene JG, Porter RHP, Eller RV and Greenamyre JT (1993) Inhibition of succinate dehydrogenase by malonic acid produces an excitotoxic lesion in rat stratum. J. Neurochem. 61: 1151-1154
Hara H, Friedlander RM, Gagliardini V, Ayata C, Fink K, Huang Z, Shimizu-Sasamata M, Yuan J and Moskowitz MA (1997) Inhibition of interleukin 1β converting enzyme family proteases reduces ischemic and excitotoxic neuronal damage. Proc. Natl. Acad. Sci. USA 94: 2007-2012
Harvey NL, Butt AJ and Kumar S (1997) Functional activation of Nedd2/ICH-1 (Caspase-2) is an early process in apoptosis J Biol Chem. 272: 13134-13139
Kane DJ, Sarafian TA, Anton R, Hahn H, Butler Gralla E, Selverstone Valentine J, Örd T and Bredesen DE (1993) Bcl-2 inhibition of neuronal death: decreased generation of reactive oxygen species. Science 262: 1274-1277
Kinoshita M, Tomimoto H, Kinoshita A, Kumar S and Noda M (1997) Up-regulation of the Nedd2 Gene encoding an ICE/CED-3-like cysteine protease in the gerbil brain after transient global ischemia. J. Cereb. Blood Flow Metab. 17: 507-514
Krajewska M, Wang H-G, Krajewski S, Zapata JM Shabaik A, Gascoyne R and Reed JC (1997) Immunohistochemical analysis of in vivo patterns of expression of CPP32 (Caspase-3), a cell death protease Cancer Res. 57: 1605-1613
Kuida K, Zheng TS, Na SQ, Kuan CY, Yang D, Karasuyama H, Rakic P and Flavell RA (1996) Decreased apoptosis in the brain and premature lethality in CPP32 deficient mice. Nature 384: 368-372
Kumar S, Kinoshita M, Noda M, Copeland NG and Jenkins NA (1994) Induction of apoptosis by the mouse Nedd2 gene, which encodes a protein similar to the product of the Caenorhabditis elegans cell death gene ced-3and the mammalian IL-1β-converting enzyme. Genes Dev. 8: 1613-1626
Li P, Allen H, Banerjee S, Franklin S, Herzog L, Johnston C, McDowell J, Paskind M, Rodman L, Salfeld J, Towne E, Tracey D, Wardwell S, Wei F-Y, Wong W, Kamen R and Seshardi T (1995a) Mice deficient in IL-1β-converting enzyme are defective in production of mature IL-1β and resistant to endotoxic shock. Cell 80: 401-414
Li Y, Sharov VG, Jiang N, Yao F, Zaloga C, Sabbah HN and Chopp M (1995b) Ultrastructural and light microscopic evidence of apoptosis after middle cerebral artery occlusion in rat. Am. J. Pathol 146: 1045-1051
Martinou J-C, Dubois-Dauphin M, Staple JK, Rodriguez I, Frankowski H, Missotten M, Albertini P, Talabot D, Catsicas S, Pietra C and Huarte J (1994) Overexpression of 8CL-2 in transgenic mice protects neurons from naturally occuring cell death and experimental ischemia. Neuron 13: 1017-1030
Myers KM, Fiskum G, Liu Y, Simmens SJ, Bredesen DE and Murphy AN (1995) Bcl-2 protects neural cells from cyanide/aglycemia-induced lipid oxidation, mitochondrial injury, and loss of viability. J Neurochem 65 2432-2440
Relton JK and Rothwell NJ (1992) Interleukin-1 receptor antagonists inhibit ischaemic and excitotoxic neuronal damage in the rat. Brain Res. Bull. 29:243-246
Schulz JB, Henshaw DR, Siwek D, Jenkins BG, Ferrante RJ, Cipolloni PB, Kowall NW. Rosen BR and Beal MF (1995a) Involvement of free radicals in excitotoxicity in vivo J Neurochem 64: 2239-2247
Schulz JB. Matthews RT, Jenkins BG, Brar P and Beal MF (1995b) Improved therapeutic window for treatment of histotoxic hypoxia with a free radical spin trap J. Cereb. Blood Flow Metab. 15: 948-952
Schulz JB, Matthews RT, Jenkins BG, Ferrante RJ, Siwek D, Henshaw DR, Cipolloni PB, Mecocci P, Kowall NW, Rosen BR and Beal MF (1995c) Blockade of neuronal nitric oxide synthase protects against excitotoxicity in vivo. J. Neurosci. 15: 8419-8429
Schulz JB, Bremen D, Reed JC, Lommatzsch J, Takayama S, Wüllner U, Lôschmann PA, Klockgether T and Weller M (1997) Cooperative interception of neuronal apoptosis by BCL-2 and BAG-1 expression Prevention of caspase activation and reduced production of reactive oxygen species. J. Neurochem 69:2075-2086
Shimizu S, Eguchi Y, Kamiike W, Wagun S, Uchiyama Y, Matsuda H and Tsujimoto Y (1996) Retardation of chemical hypoxia-induced necrotic cell death by Bcl-2 and ICE inhibitors: possible involvement of common mediators in apoptotic and necrotic signal transduction. Oncogene 12: 2045-2050
Snnivasan A, Foster LM, Testa M-P, Örd T, Keane RW, Bredesen DE and Kayalar C (1996) Bcl-2 expression in neural cells blocks activation of ICE/CED-3 family proteases during apoptosis. J. Neurosci. 16:5654-5660
Talanian RV, Quinlan C, Trautz S, Hackett MC, Mankovich JA, Banach D, Ghayur T, Brady KD and Wong WW (1997) Substrate specificities of caspase family proteases. J. Biol. Chem 272: 9677-9682
Troy CM, Stefanis L, Greene LA and Shelanski ML (1997) Nedd2 is required for apoptosis after trophic factor withdrawal, but not superoxide dismutase (SOD1) downregulation, in sympathetic neurons and PC12 cells. J. Neurosci 17 1911-1918
