Caffeine modulates CREB-dependent gene expression in developing cortical neurons

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Abstract

The Ca2+/cAMP response element binding protein CREB mediates transcription of genes essential for the development and function of the central nervous system. Here we investigated the ability of caffeine to stimulate CREB-dependent gene transcription in primary cultures of developing mouse cortical neurons. Using the CREB-dependent reporter gene CRE-luciferase we show that stimulation of CREB activity by caffeine exhibits a bell-shaped dose–response curve. Maximal stimulation occurred at 10 mM caffeine, which is known to release Ca2+ from ryanodine sensitive internal stores. In our immature neuronal cultures, 10 mM caffeine was more effective at stimulating CREB activity than depolarization with high extracellular KCl (50 mM). Quantitative real-time PCR analysis demonstrated that transcripts derived from endogenous CREB target genes, such as the gene encoding brain-derived neurotrophic factor BDNF, are increased following caffeine treatment. The dose–response curves of CREB target genes to caffeine exhibited gene-specificity, highlighting the importance of promoter structure in shaping genomic responses to Ca2+ signaling. In the presence of a weak depolarizing stimulus (10 mM KCl), concentrations of caffeine relevant for premature infants undergoing caffeine treatment increased CRE-luciferase activity and Bdnf transcript levels. The ability of caffeine to enhance activity-dependent Bdnf expression may contribute to the neurological benefit observed in infants receiving caffeine treatment.

Introduction

Caffeine, which is present in coffee, tea, soft drinks, and chocolate, is the most commonly used psychostimulant in the world [1]. Caffeine exerts multiple effects on cells. At the low doses of caffeine achieved by dietary intake (1–10 μM), the primary effect of caffeine in the central nervous system is believed to be inhibition of adenosine receptors and subsequent modulation of neurotransmitter release [2], [3]. But higher doses of caffeine can also (1) block GABA-A receptors, reducing the inhibitory input in functional neuronal networks, (2) inhibit phosphodiesterase activity leading to increased cellular cAMP levels and (3) release Ca2+ from intracellular ryanodine sensitive stores stimulating Ca2+ signaling in numerous cell types including neurons [4].

Caffeine is one of the most commonly prescribed drugs in pediatric emergency rooms. Clinically, caffeine is utilized for the treatment of premature infants with apnea [5], [6]. In newborns, the half-life of caffeine slows from the 2–5 h reported in adults to approximately 80 h in full-term infants and over 100 h in premature infants [7], [8], [9]. Due to their reduced metabolism of caffeine, premature infants receiving caffeine treatment accumulate significantly higher concentrations of plasma caffeine than those observed in adults due to dietary caffeine intake. A study in 1996 monitoring caffeine levels in the serum of 59 premature infants, however, reported a mean serum caffeine concentration of 29.9 mg/L (154 μM) with a high concentration of 93.3 mg/L observed (480 μM) [10]. More recent studies have reported serum concentrations ranging from 19 to 80 mg/L (98–412 μM) [9] or 11 to 33 mg/L (57–170 μM) [11]. Interestingly, infants treated with caffeine are less likely to exhibit neurodevelopmental deficits [12], [13], suggesting that caffeine treatment exerts a positive influence on developing neurons.

The Ca2+/cAMP response element binding protein CREB mediates transcription of genes critical for development and function of the nervous system [14]. CREB-mediated transcription of the Bdnf gene, which encodes brain-derived neurotrophic factor, promotes neuron survival, neurite outgrowth and synaptic plasticity [15]. In this study we directly test the ability of caffeine to regulate CREB activity in developing cortical neurons.

Section snippets

Cell culture and transfection

Cortical neuron cultures were prepared from mouse brains on embryonic day 15.5 (E15.5) as previously described [16]. Neurons were plated onto p35 dishes or 24-well dishes coated with 15 μg/ml polyornithine (Sigma) and 2 μg/ml laminin (Invitrogen) and cultured in Neurobasal media supplemented with 2% B27, 1 mM glutamine and penicillin/streptomycin (Invitrogen). Neurons were transiently transfected 3–5 DIV using calcium phosphate as previously described [17].

Luciferase assays

Plasmids used for luciferase assays were

Caffeine regulates CREB-dependent reporter gene expression

Increases in intracellular Ca2+ activate CREB, which stimulates gene transcription via binding to Ca2+/cAMP response element CRE in the promoter of CREB-dependent genes [18]. We assayed the ability of caffeine to stimulate CREB activity using a CREB-dependent reporter gene consisting of four tandem CREs (Stratagene). Primary cortical neurons isolated from E15.5 mouse embryos were transiently transfected with the CRE-luciferase reporter construct, along with the TK-Renilla luciferase control

Discussion

Previous studies administering caffeine in rodents have found that acute treatment with high doses of caffeine (75 mg/kg) stimulated c-fos expression in the brain [25], [26]. In contrast, lower doses aimed at mimicking dietary intake in humans (10 mg/kg) did not increase c-fos immunostaining. Since c-fos transcription can be stimulated by CREB activation, we sought to test the ability of caffeine to trigger CREB-mediated transcription in neurons. In cultured cortical neurons, we were unable to

Acknowledgments

We thank C. Roby for technical assistance with cortical neuron preparations and B. Krueger for valuable discussions. T.K. was funded as a BIRCWH Scholar for the Maryland’s Organized Research Effort in Women’s Health funded by NICHD/ORWH/NIDDK Grant K12HD43489. S. Connolly was supported by the National Institute of Health Grant NS058464.

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