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, 397 (2), 152-6

Caffeine Modulates CREB-dependent Gene Expression in Developing Cortical Neurons

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Caffeine Modulates CREB-dependent Gene Expression in Developing Cortical Neurons

Sean Connolly et al. Biochem Biophys Res Commun.

Abstract

The Ca(2+)/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 10mM caffeine, which is known to release Ca(2+) from ryanodine sensitive internal stores. In our immature neuronal cultures, 10mM caffeine was more effective at stimulating CREB activity than depolarization with high extracellular KCl (50mM). 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 Ca(2+) signaling. In the presence of a weak depolarizing stimulus (10mM 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.

Figures

Figure 1
Figure 1. Caffeine stimulates CRE-dependent transcription
Cortical neurons were transfected with CRE-luciferase to assay CREB activity and TK Renilla luciferase for normalization. Two days after transfection, neurons were stimulated with increasing concentrations of caffeine alone (A) or in combination with depolarization triggered by the addition of either 50 mM (B) or 10 mM (C) extracellular KCl. Following 5 hours of stimulation, neurons were harvested for dual-luciferase assays. Data shown is plotted relative to untreated neurons. Bars are the average of three independent transfections with standard error indicated.
Figure 2
Figure 2. Caffeine stimulates expression of endogenous CREB-dependent genes
Cortical neurons were stimulated with caffeine in the presence or absence of 10 mM KCl and harvested for RNA after 5 hours of stimulation. Real-time PCR analysis was conducted to determine the relative expression levels of A) c-fos, B) Bdnf promoter I or C) Bdnf promoter IV transcripts. RNA samples are normalized to actin amplification. Data are plotted relative to unstimulated neurons. Data shown are average of three independent experiments with standard error indicated.
Figure 3
Figure 3. Clinically relevant caffeine doses enhance Bdnf expression
Cortical neurons were stimulated with either 0.25 mM or 0.5 mM caffeine in the presence of 10 mM extracellular KCl. RNA was purified and subjected to real-time PCR analysis to quantify Bdnf promoter I, Bdnf promoter IV and TrkB transcript levels. Data plotted relative to neurons treated with 10 mM KCl alone. Data shown are average of quadruplicates with standard error indicated. Stimulation of Bdnf transcripts was statistically significant at both 250 μM and 500 μM caffeine. Increased expression of TrkB transcripts was statistically significant at 500 μM caffeine as assessed by student paired t-test (p < 0.05).

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