6533b82cfe1ef96bd128ed85

RESEARCH PRODUCT

Suppression of ischemia-induced fos expression and AP-1 activity by an antisense oligodeoxynucleotide to c-fos mRNA.

subject

description

The molecular events of brain adaptation to injury that may underlie functional recovery after stroke remain largely undefined. Recent observations of altered gene expression in ischemic brain using animal stroke models have opened new avenues for exploration of the biochemical cascades after stroke [1–11]. These postischemic events include an increase in extracellular excitatory amino acid neurotransmitters such as glutamate. Glutamate receptor–mediated activation of phospholipases and protein kinases results in the alteration of nuclear regulatory processes, including the expression of immediate early genes such as c-fos, junB, and c-jun [5, 12]. The Fos, Jun, and JunB proteins have been shown to form activator protein 1 (AP-1) through a conserved dimerization domain, i.e., the leucine zipper [13]. Transcription regulator AP-1 protein binds a specific DNA motif and is believed to transactivate the expression of a number of late effector genes [14–19]. In the ischemic brain, we have previously demonstrated an increase in AP-1 binding activity [9]. A number of genes that bear neurotrophic properties may be regulated by transcription regulator AP-1. These genes, such as heat shock protein [1, 4, 19–22], amyloid [23], neurotrophins [7], and protein tyrosine kinase receptor trkB [24], have been shown to be induced following cerebral ischemia. The causal relationship between the Fos/Jun–AP-1 cascade and the subsequent expression of the late effector genes, however, has not been studied. In an attempt to characterize the pathophysiological significance of c-fos expression following focal cerebral ischemia, we developed an antisense strategy that may suppress post-ischemic c-fos expression and consequently AP-1 binding activity. Naturally occurring antisenseRNAs are known to regulate the plasmid copy number in bacteria. The discovery of the antisense-RNA regulatory mechanism has raised the possibility of the synthesis of antisense oligonucleotides to regulate selected genes in single cells or whole animals [25–27]. The oligonucleotides employed in the antisense strategies are usually singlestranded DNA, 15 to 30 nucleotides in length. Anti-sense oligodeoxynucleotides have been used to suppress the expression of c-fos gene in cell culture systems [28–34] and in an animal model [35]. The antisense oligodeoxynucleotides are designed to block selected events such as transcription, splicing, or translation of the target mRNA. In special circumstances, similar to the delivery of cloned genes in plasmid, anti-sense oligodeoxynucleotides longer than 30 nucleotides can be internalized with the aid of lipofectin [36–38]. Although the cellular uptake of antisense oligodeoxynucleotides can be mediated via carrier-mediated endocytosis [39, 40], phosphorothioated oligodeoxynucleotide-lipofectin uptake by cells does not appear to involve endosomes or lysosomes [41, 42]. The present study of an in vivo antisense strategy utilizes a focal cerebral ischemia model in rats, in which the expression of c-fos and Fos protein and the enhanced AP-1 binding activity have been established and characterized [6, 8, 9].