Elsevier

Brain Research

Volume 898, Issue 2, 20 April 2001, Pages 224-231
Brain Research

Research report
Cyclic AMP-mediated signaling components are upregulated in the prefrontal cortex of depressed suicide victims

https://doi.org/10.1016/S0006-8993(01)02188-6Get rights and content

Abstract

The components of cyclic AMP signaling cascade (catalytic (Cα) subunit of cyclic AMP-dependent protein kinase (PKA) and cyclic AMP response element binding protein (CREB)) were quantitated by Western blotting in the prefrontal cortex of depressed suicide victims (n=23) and their matched controls (n=14). There was a significant increase in the levels of CREB, both in total (tCREB; 121±8% (mean±S.E.M.), P<0.02) and phosphorylated (pCREB; 128±9%, P<0.01) forms, but not in PKA Cα levels (109±9%, ns), in brains of depressed suicides compared to those in control subjects. The increases in CREB were specifically observed in antidepressant drug-free subjects (tCREB: 137±11%, P<0.01; pCREB: 136±12%, P<0.02; n=9), but not in the antidepressant-treated subjects (tCREB: 108±18%, ns; pCREB: 111±17%, ns; n=8). There were significant correlations between the levels of PKA and those of tCREB and pCREB in the prefrontal cortex of depressed suicides. These results indicate that the components of cyclic AMP signaling are upregulated in a coordinated manner in brains of depressed suicides and that this alteration is not related to antidepressant treatment.

Introduction

Several studies have indicated that monoaminergic neural transmission is implicated in the mechanism of action of antidepressants as well as in the pathophysiology of mood disorders [1], [6], [23]. Many markers of monoaminergic neurotransmitter signaling cascades have been shown to be altered following chronic administration of antidepressants at multiple levels. These include several classes of cell-surface receptors, adenylate cyclase and inositol phosphate second messenger systems, and the guanine nucleotide-binding regulatory (G) proteins that couple these receptors and effectors. In spite of abundant research work, however, the primary or critical site of action of antidepressants still remains obscure [1], [6], [23]. In addition, there have been limited studies on possible abnormalities in intracellular signal transduction systems beyond the receptors in the human brain [2], [4], [5], [8], [12], [20].

The cyclic AMP-mediated pathway is one of the major intracellular signal transduction systems regulated by many monoamine neurotransmitter receptors, and thus of particular interest as a potential common target for diverse antidepressant treatments and as a possible fundamental basis of depressive disorders [6]. The physiological effects of cyclic AMP are mediated by the phosphorylation of specific proteins via activation of cyclic AMP-dependent protein kinase (PKA). The inactive PKA tetramer is composed of two catalytic (C) subunits and a dimer of regulatory (R) subunits [24]. Molecular biological studies have indicated the existence of multiple isoforms of both R (RIα, RIβ, RIIα, and RIIβ) and C (Cα, Cβ, and Cγ) subunits. Binding of cyclic AMP to each R subunit of the PKA holoenzyme results in the release of two monomeric C subunits from the dimer of the R subunits, leading to the phosphorylation of substrate proteins such as cyclic AMP response element binding protein (CREB) by the free C subunits. When the levels of cyclic AMP decline, the R subunits regain the high affinity for C subunits to reform the holoenzyme. CREB is a nuclear transcription factor involved in the regulation of genes encoding several important proteins for neuronal function and plasticity, thereby playing a pivotal role in linking immediate postsynaptic events with long-term neuronal changes which are supposed to underlie the molecular and cellular alterations in depressive disorders [6].

Recently, Dowlatshahi et al. [4] reported that CREB levels in postmortem temporal cortex were lower in antidepressant-free patients with major depressive disorder relative to the CREB levels found in antidepressant-treated depressed patients and nonpsychiatric control subjects. This result was apparently in good agreement with animal studies demonstrating that chronic antidepressant treatments increase the expression of CREB in rat hippocampus [17]. However, only total levels of CREB (tCREB) were determined in these studies [4], [5]. Because CREB only becomes an active form when phosphorylated at Serine 133 [9], it is important to determine whether CREB levels in the phosphorylated form (pCREB) are altered in postmortem brains of depressed suicide victims.

In the present study, we have measured tCREB and pCREB by immunoblotting techniques, using specific antibodies, in postmortem brains from drug-free depressed suicides, antidepressant-treated depressed suicides, and control subjects. Moreover, the levels of PKA C subunit, one of the main activators of CREB, were also determined in the same sample to get a better understanding regarding the relationship among these proteins in the human brain.

Section snippets

Postmortem brain samples and tissue preparation

Human brains were obtained at autopsy from the Institute of Forensic Medicine, Geneva, Switzerland. This study was approved by the research and ethics review board of the Department of Psychiatry, Faculty of Medicine, University of Geneva. The specimens of postmortem human brain were obtained following all the legal procedures of the République et Canton de Genève, which, in some cases, included assent from the families.

Specimens of the right prefrontal cortex (Brodmann area 9) were collected

Effects of PMD and age on target protein immunoreactivities

Although the PMD and age were matched as carefully as possible and there were no differences between control subjects and depressed suicide victims (see Table 1), the influence of both parameters on the immunoreactivities of PKA Cα, tCREB, and pCREB were examined in the prefrontal cortex of 14 male control subjects with PMD of 3–81 h and with ages of 15–52 years. As shown in Fig. 1, all three proteins were readily detected regardless of PMD of the subjects, and no statistically significant

Discussion

In the present investigation, we have quantitated and compared the immunoreactivities of PKA Cα, the major catalytic isoform of PKA in most mammalian tissues [22], [25], and CREB, the downstream main target protein of PKA [3], [11], in the prefrontal cortex of depressed suicide victims and matched control subjects. With regard to CREB, phosphorylated and total immunoreactivities were determined to assess its functional relevance, because it has been shown that CREB is activated by

Acknowledgements

This study was supported by grants 31-52242.97 from FNSRS, Switzerland and 09670970 from the Ministry of Education, Science and Culture of Japan. Y.O. was a recipient of the Naito Foundation. The authors are grateful to Dr. Megumi Yamamoto for her helpful advice on the method of immunoblotting of CREB. J.A.G.-S. is a member of the Institut d’Estudis Catalans (Barcelona, Spain).

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