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. 2015 Oct 27;5(10):e667.
doi: 10.1038/tp.2015.158.

Transcriptomic and Genetic Studies Identify NFAT5 as a Candidate Gene for Cocaine Dependence

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Free PMC article

Transcriptomic and Genetic Studies Identify NFAT5 as a Candidate Gene for Cocaine Dependence

N Fernàndez-Castillo et al. Transl Psychiatry. .
Free PMC article

Abstract

Cocaine reward and reinforcing effects are mediated mainly by dopaminergic neurotransmission. In this study, we aimed at evaluating gene expression changes induced by acute cocaine exposure on SH-SY5Y-differentiated cells, which have been widely used as a dopaminergic neuronal model. Expression changes and a concomitant increase in neuronal activity were observed after a 5 μM cocaine exposure, whereas no changes in gene expression or in neuronal activity took place at 1 μM cocaine. Changes in gene expression were identified in a total of 756 genes, mainly related to regulation of transcription and gene expression, cell cycle, adhesion and cell projection, as well as mitogen-activeated protein kinase (MAPK), CREB, neurotrophin and neuregulin signaling pathways. Some genes displaying altered expression were subsequently targeted with predicted functional single-nucleotide polymorphisms (SNPs) in a case-control association study in a sample of 806 cocaine-dependent patients and 817 controls. This study highlighted associations between cocaine dependence and five SNPs predicted to alter microRNA binding at the 3'-untranslated region of the NFAT5 gene. The association of SNP rs1437134 with cocaine dependence survived the Bonferroni correction for multiple testing. A functional effect was confirmed for this variant by a luciferase reporter assay, with lower expression observed for the rs1437134G allele, which was more pronounced in the presence of hsa-miR-509. However, brain volumes in regions of relevance to addiction, as assessed with magnetic resonance imaging, did not correlate with NFAT5 variation. These results suggest that the NFAT5 gene, which is upregulated a few hours after cocaine exposure, may be involved in the genetic predisposition to cocaine dependence.

Conflict of interest statement

BF received a speaker fee from Merz. The remaining authors declare no conflict of interest for this research.

Figures

Figure 1
Figure 1
Gene expression changes caused by exposure to 5 μM cocaine in vitro after 6 h. (a) Representative over-represented biological categories (Gene Ontology terms, GO) and pathways (Kyoto Encyclopedia of Genes and Genomes, KEGG) identified by DAVID software among the differentially expressed genes. The number of genes with altered expression included in each category is indicated on the right side of the bar. (b) Gene network involved in molecular transport, cell-to-cell signaling and interaction and cellular development (score=34). The green and red nodes in the pathway indicate the down- and upregulated genes, respectively, induced by 5 μM cocaine–HCl after 6 h.
Figure 2
Figure 2
Quantitative real-time PCR validation of gene expression changes identified by microarray experiments. Transcription levels of seven genes involved in neuronal adaptations and transcription were determined by qRT-PCR at different time points after a 30-min exposure to 5 μM cocaine. Significant differences compared with control cells (not exposed to cocaine) normalized to GAPDH are indicated (*P<0.05). Error bars indicate s.d.
Figure 3
Figure 3
Neuronal activity changes induced by cocaine. (a) Comparison of a phase-contrast snapshot (top) with the fluorescence equivalent (bottom). For the latter, bright spots are firing neurons, showing the ability to track neuronal activity with single-cell resolution. The arrowhead indicates the same neuron in both images. (b) Representative fluorescence traces of neuronal activity for gradual exposure to higher cocaine concentrations. Neuronal response is weak for 1 μM concentration, and increases progressively for 5 and 10 μM. (c) Detail of neuronal response to cocaine, showing that the firing amplitude increases with cocaine concentration. Each trace at a given concentration is an average over the responses of 10 different neurons. (d) Comparison of the fraction of active neurons, showing that network activity significantly increases for 5 and 10 μM cocaine exposure compared to the 0 μM (spontaneous acitivity) and 1 μM concentration. (e) Average number of activations per neuron for the different conditions, highlighting the much higher neuronal activity at 5 and 10 μM cocaine. Significant differences compared to 0 μM cocaine are indicated. *P<0.05 and **P<0.01. Error bars indicate s.d.
Figure 4
Figure 4
Effect of rs1437134 on gene expression. Effect on gene expression of the two alleles of the associated variant rs1437134 in absence and presence of hsa-miR-509 in HeLa cells (a) and in SH-SY5Y cells (b). Relative luciferase expression of the pmirGlo vector (control) and the constructs with the 3′-untranslated region of NFAT5 containing the rs1437134 A and G alleles. Significant differences between the G and the A allele are indicated as *P<0.05 and **P<0.01. Error bars indicate s.d.

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