Microarray Analysis of Ethanol-Treated Cortical Neurons Reveals Disruption of Genes Related to the Ubiquitin-Proteasome Pathway and Protein Synthesis Ramana Gutala, Ju Wang, Sheila Kadapakkam, Yoon Hwang, Maharaj Ticku, and Ming D. Li 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 - and -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 de- creased 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. Key Words: Ethanol, Ubiquitin-Proteasome Pathway, Ribosomal Proteins, Cortical Neurons, cDNA Microarray. I N WESTERN SOCIETIES, alcohol addiction is a prev- alent form of drug abuse. Behavioral responses to chronic alcohol intake lead to tolerance, dependence, re- inforcement, sensitization, and craving (Mayfield et al., 2002). Chronic exposure to alcohol results in insidious damage to several body organs, especially the brain, heart, and liver. Furthermore, chronic alcohol abuse is associated with increased morbidity and mortality (Marshall et al., 1994). Addiction to alcohol and related behaviors are gov- erned by a complex interplay of genetic and environmental factors (Enoch and Goldman, 2001). Identification of ethanol-responsive genes or pathways would enhance our understanding of ethanol action at the cellular level and would have implications for pharmacological interventions for alcohol addiction. Ethanol modulates the expression levels of several neu- rotransmitter receptors, such as receptors of NMDA (Ku- mari et al., 2003), -aminobutyric acid (Davies, 2003), nic- otinic acetylcholine (Zuo et al., 2001), AMPA (Bruckner et al., 1997), and serotonin (Nevo et al., 1995). In cortical neurons, acute ethanol exposure inhibits NMDA receptor function, whereas chronic ethanol treatment increases the NMDA receptor number and function, with a concomitant increase in NMDA receptor polypeptide subunits and pro- tein levels (Bao et al., 2001; Kumari and Ticku, 2000). From the Departments of Psychiatry (RG, JW, YH, MDL) and Pharma- cology (SK, MT, MDL), The University of Texas Health Science Center at San Antonio, San Antonio, Texas. RG and JW contributed equally to the work. Received for publication April 5, 2004; accepted September 28, 2004. Supported by NIH Grants DA-13783 and DA-12844 (MDL). Reprint requests: Ming D. Li, PhD, Mail Code 7792, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Dr., San Antonio, TX 78229; Fax: 210-567-0853; E-mail: lim2@uthscsa.edu. Copyright © 2004 by the Research Society on Alcoholism. DOI: 10.1097/01.ALC.0000148117.17707.B4 0145-6008/04/2812-1779$03.00/0 ALCOHOLISM:CLINICAL AND EXPERIMENTAL RESEARCH Vol. 28, No. 12 December 2004 Alcohol Clin Exp Res, Vol 28, No 12, 2004: pp 1779–1788 1779