Background: Chronic ethanol abuse results in deleterious behavioral responses such as tolerance, dependence, reinforcement, sensitization, and craving. The objective of this research was to identify transcripts that are differentially regulated in ethanol-treated cortical neurons compared with controls by using a pathway-focused complementary DNA microarray.
Methods: Cortical neurons were isolated from postconception day 14 C57BL/6 mouse fetuses and cultured according to a standard protocol. The cortical neuronal cells were treated with 100 mM ethanol for five consecutive days with a change of media every day. A homeostatic pathway-focused microarray consisting of 638 sequence-verified genes was used to measure transcripts differentially regulated in four ethanol-treated cortical neuron samples and four control samples. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis was used to verify the mRNA expression levels of genes of interest detected from the microarray experiments.
Results: We identified 56 down-regulated and 10 up-regulated genes in ethanol-treated cortical neurons relative to untreated controls at a 5% false-discovery rate. The expression of many genes involved in ubiquitin-proteasome and protein synthesis was decreased by ethanol, including ubiquitin B, ubiquitin-like 3, ubiquitin-conjugating enzyme E3A, 20S proteasome alpha- and beta-subunits, and members of the ribosomal proteins. Furthermore, the mRNA expression of heat shock proteins, myristoylated alanine-rich protein kinase C substrate, phosphatase and tensin homolog deleted on chromosome 10, and FK506 binding protein rapamycin-associated protein (FKBP) (mTOR) was also decreased in ethanol-treated cortical neurons. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis of genes involved in the ubiquitin-proteasome cascade revealed a down-regulation of these genes, thereby corroborating our microarray results.
Conclusions: Our results indicate that chronic ethanol treatment of cortical neurons resulted in decreased mRNA expression of genes involving the ubiquitin-proteasome pathway and ribosomal proteins together with mTOR expression leading to disruption of protein degradation mechanism and impairment of protein synthesis machinery.