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Published on: July 30, 2013
Cocaine exposure at a sublethal concentration downregulates CREB functions in cultured neuroblastoma cells
Mei-Jiang Feng1, Shu-E Yan, Qing-Shan Yan
1Department of Biomedical and Therapeutic Sciences, University of Illinois College of Medicine at Peoria, Peoria, IL 61656, USA.
Abstract:
Previous studies showed that prenatal cocaine in an animal model decreased brain-derived neurotrophic factor (BDNF) activity in offspring's brain. Since BDNF is one of target genes of cAMP response element-binding protein (CREB), this study examined effects of cocaine on CREB activities in a human neuroblastoma (SK-N-AS) cell line. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazodium bromide) assay indicated that cocaine exposure at 5 microM for 24 h had no significant influences on cell viability. However, a 24-h exposure to cocaine at the same concentration significantly decreased the level of phosphorylated CREB, although no significant changes in total CREB proteins were observed. Consistent with reduced CREB phosphorylation, the electrophoretic mobility shift assay showed that exposure to 5 microM of cocaine for 24 h also inhibited CREB binding activity and significantly decreased BDNF mRNA expression. In addition, exposure to 5 microM cocaine for 24 h attenuated the glutamic acid-evoked increase in the intracellular Ca2+ concentration. Taken together, these findings suggest that cocaine exposure at the sublethal concentration downregulates CREB functions in the cultured SK-N-AS cell line, and that diminished intracellular Ca2+ responses may be associated in part with cocaine-induced downregulation of CREB activity.
Insights
Cocaine exposure in a human neuroblastoma cell line reduced phosphorylated CREB and brain-derived neurotrophic factor (BDNF) mRNA. This suggests cocaine may impair CREB function and BDNF expression, potentially impacting neuronal development.
Area of Science:
- Neuroscience
- Molecular Biology
- Pharmacology
Background:
- Prenatal cocaine exposure in animal models is linked to decreased brain-derived neurotrophic factor (BDNF) activity.
- Brain-derived neurotrophic factor (BDNF) is a key neurotrophin involved in neuronal survival, growth, and plasticity.
- BDNF is regulated by the cAMP response element-binding protein (CREB), a transcription factor crucial for gene expression.
Purpose of the Study:
- To investigate the effects of cocaine on CREB activity and BDNF expression in a human neuroblastoma cell line (SK-N-AS).
- To determine if cocaine exposure impacts CREB phosphorylation, DNA binding activity, and BDNF mRNA levels.
- To explore the relationship between cocaine exposure, CREB function, and intracellular calcium signaling.
Main Methods:
- Human neuroblastoma SK-N-AS cells were exposed to cocaine (5 microM) for 24 hours.
- Cell viability was assessed using the MTT assay.
- CREB phosphorylation and total CREB protein levels were measured.
- CREB binding activity was analyzed using electrophoretic mobility shift assay (EMSA).
- BDNF mRNA expression was quantified.
- Glutamic acid-evoked intracellular Ca2+ concentration changes were monitored.
Main Results:
- Cocaine exposure at 5 microM for 24 hours did not affect cell viability.
- Cocaine significantly decreased phosphorylated CREB levels without altering total CREB protein.
- Cocaine inhibited CREB binding activity and reduced BDNF mRNA expression.
- Cocaine exposure attenuated the glutamic acid-induced increase in intracellular Ca2+ concentration.
Conclusions:
- Cocaine exposure at sublethal concentrations downregulates CREB function in cultured human neuroblastoma cells.
- The observed decrease in CREB activity is associated with reduced BDNF mRNA expression.
- Diminished intracellular Ca2+ responses may be partly linked to cocaine-induced CREB downregulation.
