IMMUNOHISTOCHEMICAL AND BIOCHEMICAL ASSESSMENT OF CASPASE-3 ACTIVATION AND DNA FRAGMENTATION FOLLOWING TRANSIENT FOCAL ISCHEMIA IN THE RAT M. A. DAVOLI, 1 J. FOURTOUNIS, 1 J. TAM, S. XANTHOUDAKIS, D. NICHOLSON, G. S. ROBERTSON, G. Y. K. NG and D. XU Ã Merck Frosst Center for Therapeutic Research, P.O. Box 1005, Pointe Claire-Dorval, QC, Canada H9R 4P8 AbstractöIn the present study, we evaluated the time-course of caspase-3 activation, and the evolution of cell death following focal cerebral ischemia produced by transient middle cerebral artery occlusion in rats. Ischemia-induced active caspase-3 immunoreactivity in the striatum but not the cortex at 3 and 6 h time points post-reperfusion. Furthermore, using a novel approach to visualize enzymatic activity, vC-APP, a C-terminal cleavage product of APP generated by caspase-3, was found to immunolocalize to the same areas as active caspase-3. Double-labeling studies demonstrated co- localization of these two proteins at the cellular level. Further double-labeling experiments revealed that active caspase-3 was con¢ned to neuronal cells which were still viable and thus immunoreactive for NeuN. DNA fragmentation, assessed histologically by terminal dUTP nick-end labeling (TUNEL), was observed in a small number of cells in the striatum as early as 3 h, but only began to appear in the cortex by 6 h. DNA fragmentation was progressive, and by 24 h post- reperfusion, large portions of both the striatum and cortex showed TUNEL positive cells. However, double-labeling of active caspase-3 with TUNEL showed only minimal co-localization at all time-points. Thus, caspase-3 activation is an event that appears to occur prior to DNA fragmentation. As a con¢rmation of the histological TUNEL data, 24 h ischemia also induced the generation of nucleosome fragments, evidenced by cell death enzyme-linked immunosorbent assay. Using a novel ischemia-induced substrate cleavage biochemical approach, spectrin P120 fragment, a caspase- speci¢c cleavage product of alpha II spectrin, a cytoskeletal protein, was shown to be elevated by western blotting. Brain concentrations of both nucleosomes and spectrin P120 correlate with the degree of injury previously assessed by triphenyltetrazolium chloride staining and infarct volume calculation. Together, our ¢ndings suggest a possible associ- ation between caspase-3 activation and ischemic cell death following middle cerebral artery occlusion brain injury. ß 2002 IBRO. Published by Elsevier Science Ltd. All rights reserved. Key words: apoptosis, nucleosomes, spectrin, amyloid precursor protein, infarct. Caspases are homologous cysteine proteases that exhibit a primary speci¢city to cleave after aspartic acid residues in peptide substrates, allowing for highly selective pro- teolysis in animal cells (Stennicke and Salvesen, 1998). At least 14 members of the caspase family have thus far been identi¢ed (Eldadah and Faden, 2000). Based on their preferred substrate speci¢cities (tetrapeptide motifs), which have been de¢ned using a positional scanning combinatorial library (Rano et al., 1997; Thornberry et al., 1997), these enzymes can be divided into three general groups: apoptotic initiators (caspase-8 and caspase-9), apoptotic executioners (caspase-3, -6, and -7), and in£ammatory mediators (caspase-1, -4, -5, -11, -13). There is some amount of evidence in the liter- ature linking ischemia-induced neuronal apoptosis to the caspase family of enzymes; however, their direct role is still under scrutiny (for review see Loetscher et al., 2001). The most notable association is seen in experimental models that elicit mild injury in the adult brain (Endres et al., 1998; Chen et al., 1998; Himi et al., 1998), and in neonate models of hypoxic ischemia (Pulera et al., 1998; Cheng et al., 1998; Liu et al., 1999; Nakajima et al., 2000). Caspase-3 is believed to be the main executioner pro- tease of the apoptotic cascade, as evidenced in studies of caspase-3 knockout mice that die approximately 3 weeks after birth due to massive brain anomalies including ectopic growths and hyperplasias resulting in disorgan- ized, overdeveloped structures (Kuida et al., 1996). Cas- pase-3 exists as a 32-kDa proenzyme that can be activated through catalytic cleavage by a number of pro- teases, thus yielding two smaller sub-units of 17 kDa (resulting from a subsequent autocatalytic cleavage of a 20-kDA pro-domain containing fragment) and 12 kDA. The active form of the enzyme exists as a tetrameric enzyme complex composed of two large (17-kDA) and two small (12-kDA) sub-units (Nicholson et al., 1995). 125 1 Both authors contributed equally to the work. *Corresponding author. Tel.: +1-514-428-3661; fax: +1-514-428- 3921. E-mail address: daigen_xu@merck.com (D. Xu). Abbreviations: vC-APP, C-terminal neo-epitope of APP generated by caspase cleavage; APP, amyloid precursor protein; CCA, common carotid artery; CDE, cell death ELISA; EDTA, ethyl- enediaminetetra-acetate; ELISA, enzyme-linked immunosorbent assay; FITC, £uorescein isothiocyanate; MCA, middle cerebral artery; MCAo, MCA occlusion; PBS, phosphate-bu¡ered saline; TTC, triphenyltetrazolium chloride; TUNEL, terminal dUTP nick-end labeling. NSC 5800 14-10-02 Cyaan Magenta Geel Zwart www.neuroscience-ibro.com Neuroscience Vol. 115, No. 1, pp. 125^136, 2002 ß 2002 IBRO. Published by Elsevier Science Ltd All rights reserved. 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