Pergamon Neuroscience Vol. 89, No. 2, pp. 437452, 1999 Copyright © t998 IBRO. Published by Elsevier ScienceLtd Printed in Great Britain. All rights reserved PII: S0306-4522(98)00352-2 0306-4522/99 $19.00+0.00 PERSISTENT PHOSPHORYLATION OF CYCLIC AMP RESPONSIVE ELEMENT-BINDING PROTEIN AND ACTIVATING TRANSCRIPTION FACTOR-2 TRANSCRIPTION FACTORS FOLLOWING TRANSIENT CEREBRAL ISCHEMIA IN RAT BRAIN B. R. HU,*:~ C. M. FUX,'~ M. E. MARTONE,* J. A. ZIVIN* and M. H. ELLISMAN* *Department of Neurosciences and National Center for Microscopy and Imaging Research, University of California, San Diego, CA 92093-0624, U.S.A. tDepartment of Neurobiology, Swiss Federal Institute of Technology, Honggerberg, CH-8093 Zurich, Switzerland Abstract The transcription factors cyclic AMP responsive element-binding protein (CREB) and activat- ing transcription factor-2 were studied in rat brains subjected to 15 min ischemia followed by varied periods of reperfusion using western blot and immunocytochemical analyses. The total amounts of both CREB and activating transcription factor-2 were not altered in the hippocampus after ischemia. In contrast, levels of the phosphorylated forms of both transcription factors decreased during ischemia but rebounded following reperfusion. The phospho-forms of CREB and activating transcription factor-2 showed regional and temporal differences in their expression. Phospho-CREB was increased relative to control levels at 30 min, and continued to increase for at least three days postischemia, mainly in dentate granule ceils. The level of phospho-activating transcription factor-2 appeared to be higher in CAI pyramidal cells than in dentate granule cells after ischemia. The present findings suggest that the signaling pathways for phosphorylation of CREB may be neuroprotective for dentate cells, which are relatively resistant to ischemic insults. The increased phospho-activating transcription factor-2 may reflect increased stresses in these neurons. The more modest activation of CREB pathways in CA1 neurons may not be enough to overcome the increased stresses in these neurons, contributing to delayed neuronal death. ~) 1998 IBRO. Published by Elsevier Science Ltd. Key words: transient cerebral ischemia, CREB, ATF-2, phosphorylation, hippocampus, cell death. Transient cerebral ischemia leads to cell death selec- tively in CA1 pyramidal neurons at approximately three days after the ischemic episode, while leaving CA3/dentate gyrus (DG) and most neocortical neurons intact. 26'4° This selective and delayed cell death provides a model for studying the mechanisms underlying reversible and irreversible cell damage in postischemic hippocampal neurons. The mechanisms leading to ischemic delayed neuronal death are not fully understood. A remarkable molecular alteration is the expression of a variety of genes including transcription factors, neurotrophins and neurotrophin receptors, heat-shock proteins ~To whom correspondence should be addressed. Abbreviations: ABC, avidin biotin horseradish peroxidase complex; ATF-2, activating transcription factor-2; BSA, bovine serum albumin; CREB, cyclic AMP responsive element-binding protein; DAB, 3,3'-diaminobenzidine; DG, dentate gyms; DTT, dithiothreitol; EDTA, ethylenediaminetetra-acetate; EGTA, ethyleneglycolbis- (aminoethylether)tetra-acetate; IEG, immediate-early gene; LTP, long-term potentiation; PBS, phosphate- buffered saline; RT, room temperature; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electro- phoresis. and activity-regulated proteins after transient ischemia, ls'24'25a'27'3°'32'37"38'45 Most of these genes are also expressed under physiological or other pathological conditions such as long-term poten- tiation (LTP), seizure or excitotoxin-induced cell death. 3,11,12,33,41,47 The cyclic AMP responsive element-binding pro- tein (CREB) and activating transcription factor-2 (ATF-2) belong to the same family of transcription factors (ATF/CREB). The dimerization of two tran- scription factors in the ATF/CREB family is neces- sary for the induction of transcriptional activity through a common domain called bZIP. Unlike transcription factors of the fos or jun family that are immediate-early genes (lEGs) and are regulated by synthesis of new proteins and by covalent modifi- cation, CREB and ATF-2 are constitutive proteins and are activated mainly by phosphorylation fol- lowing stimuli. 22"25 Phosphorylation of CREB on Serine-133 (Set-133) residue or of ATF-2 on the Threonine-69 (Thr-69) or -71 (Thr-71) residues promotes transcription of genes with an upstream cAMP responsive element known as CRE. 46 Two signaling pathways, i.e. protein kinase A and 437