Induction of cyclin-dependent kinase 5 in the hippocampus by chronic electroconvulsive seizures: role of [Delta]FosB

Abstract

The transcription factor DeltaFosB is induced in the hippocampus and other brain regions by repeated electroconvulsive seizures (ECS), an effective antidepressant treatment. The unusually high stability of this protein makes it an attractive candidate to mediate some of the long-lasting changes in the brain caused by ECS treatment. To understand how DeltaFosB might alter brain function, we examined the gene expression profiles in the hippocampus of inducible transgenic mice that express DeltaFosB in this brain region by the use of cDNA expression arrays that contain 588 genes. Of the 430 genes detected, 20 genes were consistently upregulated, and 14 genes were downregulated, by >50%. One of the upregulated genes is cyclin-dependent kinase 5 (cdk5). On the basis of its purported role in regulating neuronal structure, we studied directly whether cdk5 is a true target for DeltaFosB. Upregulation of cdk5 immunoreactivity in the hippocampus was confirmed by Western blotting in the DeltaFosB-expressing transgenic mice as well as in rats treated chronically with ECS. Chronic ECS treatment also increased, in the hippocampus, the phosphorylation state of tau, a microtubule-associated protein that is a known substrate for cdk5. A 1.6 kb fragment of the cdk5 promoter was cloned, and activity of the promoter was found to be increased after overexpression of DeltaFosB in cell culture. Moreover, mutation of the single consensus activator protein-1 site contained within the cdk5 promoter fragment completely abolished activation of the promoter by DeltaFosB. Together, these results suggest that cdk5 is one target by which DeltaFosB produces some of its physiological effects in the hippocampus and thereby mediates certain long-term consequences of chronic ECS treatment.

Fig. 1.

Identification of cdk5 as one of the downstream target genes for ΔFosB in the hippocampus of inducible bitransgenic mice using cDNA expression arrays. A, Schematic diagram of the tetracycline expression system used for the inducible tissue-specific expression of ΔFosB (Chen et al., 1998). Gene 1 encodes the tetracycline transactivator (tTA) under the control of the neuron-specific enolase (NSE) promoter. Gene 2 encodes ΔFosB under the control of the tetracycline-responsive promoter with seven tetracycline operators (TetOp). B, Strategy for searching downstream target genes for ΔFosB in the hippocampus of inducible ΔFosB-expressing bitransgenic mice. Total RNA was isolated from five bitransgenic mice, either expressing or not expressing ΔFosB, and pooled. Poly(A⁺) RNA was isolated from the pooled total RNA and used as a template for the synthesis of a³²P-labeled cDNA probe. The cDNA probes were hybridized to the arrays, and the arrays were analyzed by the PhosphorImager.C, Gene expression profiles of the hippocampus of the bitransgenic mice, either expressing or not expressing ΔFosB, from a portion of the resulting cDNA expression arrays. Positions of the ΔFosB and cdk5 genes are indicated by arrows. The results are representative of three independent determinations.

Fig. 2.

Upregulation of cdk5 immunoreactivity in the hippocampus of inducible bitransgenic mice after ΔFosB expression.A, A representative immunoblot shows cdk5 levels in the hippocampus of bitransgenic mice expressing (+) or not expressing (−) ΔFosB. B, Levels of cdk5 immunoreactivity are given as arbitrary OD units and are expressed as the mean ± SEM (n = 5 animals in each treatment group). *p < 0.05 by Student's ttest.

Fig. 3.

Induction of cdk5 promoter activity by ΔFosB.A, Schematic structure of a fragment of the 5′-promoter of the cdk5 gene is shown. Several putative response elements within the promoter region are indicated. The AP-1 site framedby a rectangular box and its adjacent sequences are shown. The AP-1 sequence in a mutated promoter (underlined sequence) is also shown. B, Luciferase activity was measured in a C6 glioma cell line that supports the inducible expression of ΔFosB (Chen et al., 1997) transfected with the wild-type (cdk5-luc) or mutated (mutcdk5-luc) cdk5 promoter in pGL3-basic. Data are expressed as the mean percent change in promoter activity in the presence of ΔFosB compared with that in the absence of ΔFosB (± SEM; n = 3). The results are representative of two independent replications. *p< 0.05 by Student's t test.

Fig. 4.

Upregulation of cdk5 AP-1-binding activity in the hippocampus of inducible bitransgenic mice after ΔFosB expression.A, The sequence of the cdk5 AP-1 oligonucleotide used as the probe is shown. The ³²P-labeled nucleotides are indicated by dots. B, A representative autoradiogram shows the dramatic induction of cdk5 AP-1-binding activity after ΔFosB expression. The results are representative of three independent replications.

Fig. 5.

Upregulation of cdk5 immunoreactivity in rat hippocampus by chronic ECS treatment. A,Top, A representative immunoblot shows cdk5 levels in the hippocampus after sham or ECS treatment. Bottom, Levels of cdk5 immunoreactivity are given as arbitrary OD units and are expressed as the mean ± SEM (n = 8 animals in each treatment group). B, Top, A representative autoradiogram shows cdk5 AP-1-binding activity after sham or ECS treatment. Bottom, Levels of cdk5 AP-1-binding activity are given as arbitrary OD units and are expressed as the mean ± SEM (n = 8 animals in each treatment group). *p < 0.05 by Student'st test.

Fig. 6.

Upregulation of tau phosphorylation in rat hippocampus by chronic ECS treatment. A, A representative immunoblot showing levels of phospho-tau proteins after sham or ECS treatment. B–F, Levels of phospho-tau, in arbitrary OD units, for each tau isoform ± SEM (n = 3). *p < 0.05 by Student's t test.

Fig. 7.

Regulation of p35 and p25 immunoreactivity in rat hippocampus by chronic ECS treatment. A, A representative immunoblot shows p35 and p25 levels after sham or chronic ECS treatment. B, Levels of p35 and p25 immunoreactivity are given as arbitrary OD units and are expressed as the mean ± SEM (n = 8 animals in each treatment group). *p < 0.05 by Student'st test.