Expression of the neurotrophin receptor trkB is regulated by the cAMP/CREB pathway in neurons

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Abstract

trkB as receptor for neurotrophins brain-derived neurotrophic factor (BDNF)/neurotrophin (NT)-4/5 plays a crucial role during development, maintenance of the adult brain, and its adaptation to injury or pathological conditions. In spite of this, very little is known about the mechanisms that regulate its expression. Here, we show that forskolin (Fk) rapidly stimulates the expression of both the full-length and truncated trkB isoforms in primary cultures of cortical neurons. Gel shift assays and transient transfection experiments demonstrate that this activation occurs via a protein kinase A (PKA)/cyclic AMP-responsive element-binding protein (CREB)-dependent mechanism. Activated CREB binds to the second cyclic AMP (cAMP)-responsive element (CRE) of the two CRE sites located within the P2 promoter of the trkB gene, which is able to confer cAMP responsiveness to a heterologous promoter. Our results illustrate that the trkB gene is a target for CREB regulation and explain the increase of trkB expression produced in different adaptative responses of the nervous system where CREB is participating.

Introduction

The neurotrophins are a family of secreted proteins that potently regulate diverse neuronal responses essential for the proper development and maintenance of the nervous system. Family members include nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin 4/5 (NT-4/5). Classically, they are known to serve as target-derived trophic factors that adjust the type and number of afferent synapses by promoting the survival of discrete neuronal populations. However, it is now very well established that neuroptrophins have many other functions. They regulate differentiation, influence cell fate choices, and control neurite morphology (recently reviewed in Bibel and Barde, 2000). These neurotrophins act through the Trk family of tyrosine kinase receptors. NGF is ligand for TrkA, whereas BDNF and NT-4/5 bind to the products of the trkB gene and NT-3 binds to TrkC. Besides, these neurotrophins bind to a common receptor, known as p75, with low affinity Barbacid, 1994, Bothwell, 1995, Lindsay, 1994).

The trkB gene is a large and complex locus encoding four isoforms that are generated by differential splicing: a full-length receptor containing the tyrosine kinase domain (trkB full-length) Klein et al., 1989, Klein et al., 1991, two truncated forms lacking catalytic activity (trkB-T1, trkB-T2) (Klein et al., 1990), and a novel isoform lacking the tyrosine kinase domain but containing an Shc binding site (trkB-T-Shc) (Stoilov et al., 2002). Both the full-length and the truncated isoforms of trkB are transcribed from two different promoters (Barettino et al., 1999).

Besides the traditional role in neuronal development and survival, BDNF/trkB is also required for the maintenance and function of the adult nervous system as demonstrated recently, studying heterozygote and conditional knockout animals. Young BDNF+/− adults show learning deficits and develop an enhanced aggressiveness and hyperphagia, accompanied by weight gain Kernie et al., 2000, Lyons et al., 1999. Conversely, mice made trkB-deficient in adulthood show a substantial reduction of the long-term potentiation (LTP) in the hippocampus. Moreover, these animals become increasingly impaired over time with regard to their spatial learning behavior Minichiello et al., 1999, Xu et al., 2000.

In the adult brain, trkB expression is able to respond to external stimuli. Thus, a rapid and transient increase on trkB mRNA levels is produced by cerebral hypoxic/ischemic injury Merlio et al., 1993, Narumiya et al., 1998, by repeated stress (Nibuya et al., 1999) and by the late phase of LTP in the hippocampus (Dragunow et al., 1997). Kainate-induced seizures and antidepressant drug treatments also stimulated trkB expression in brain cortex, striatum, and hippocampus Merlio et al., 1993, Nibuya et al., 1995, Salin et al., 1995. Despite this wealth of information, the factors that control trkB expression during development or in the adult brain remain mostly unknown. We have previously described that thyroid hormone represses trkB expression in the developing brain through a negative response element (Pombo et al., 2000). In addition, we have characterized the trkB promoters (Barettino et al., 1999) and analyzed putative regulatory sequences on this gene. In search for the possible regulators of the trkB gene, we found that forskolin (Fk) rapidly stimulates the expression of both the full-length and truncated trkB isoforms in primary cultures of cortical neurons. We also demonstrate that this activation occurs via a protein kinase A (PKA)/cyclic AMP-responsive element-binding protein (CREB)-dependent mechanism through a novel cyclic AMP (cAMP)-responsive element (CRE) site present in the trkB gene.

Section snippets

Forskolin stimulates trkB expression in cortical neurons

In search of possible regulators of the trkB gene, we studied if trkB mRNA expression was affected by changes in cAMP intracellular levels. RNase protection assays were performed using total RNA isolated from rat cultured cortical neurons that were stimulated with 30 μM Fk for varying periods of time, because Fk is known to produce an increase in intracellular cAMP levels (Insel and Ostrom, 2003). The riboprobe used was designed to generate two protected fragments of different size, one

Discussion

In this study, we have demonstrated that cAMP/PKA stimulates trkB gene expression through the binding of CREB to a CRE site located 450 nucleotides 5′ of the site of transcription initiation of the trkB P2. The expression of the neurotrophin receptor trkB is rapidly increased in primary cultures of rat cortical neurons after Fk treatment. Cortical neurons, as previously reported for hippocampal neurons, express predominantly the full-length trkB receptor. In these cultures, elevation of cAMP by

Cortical neuron cultures

Primary cultures of cortical neurons were prepared from brain cortices of Wistar rats embryos (E18/E19). Fetuses were obtained under aseptic conditions, and the cerebral cortices were dissected free of meninges and mechanically dissociated in 1 ml of minimum essential medium (MEM, Invitrogen) supplemented with 16 μg/ml gentamicin, 2mM glutamine, 0.6% glucose, 10% fetal bovine serum (FBS), and 10% horse serum. The dissociated neurons were plated on 100-mm plates precoated with poly-d-lysine (10

Acknowledgements

We thank Dr. D. Barettino for the first identify of the putative CREs in the trkB promoter, Dr. M. Dı́az-Guerra for carefully reading the manuscript and helpful discussion. This work was supported by grant to A.R.-P., from “Dirección General de Investigación Cientı́fica y Técnica” (Ministerio de Ciencia y Tecnologı́a: SAF2001-1624). R.D. and G.E. were supported by predoctoral fellowships from “Ministerio de Ciencia y Tecnologı́a” and “Comunidad Autónoma de Madrid,” respectively.

References (43)

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