Effects of chronic ethanol administration on brain protein levels: A proteomic investigation using 2-D DIGE system

Abstract

The effects of chronic ethanol treatment on the brain proteome were investigated in the long-fin striped strain of zebrafish Danio rerio. Prolonged exposure to 0.5% (v/v) ethanol resulted in the development of tolerance to the ethanol-induced disruption of normal swimming behavior. This behavioral tolerance was manifested after two weeks of continuous treatment and was maintained for an additional three weeks. After four weeks of ethanol treatment, zebrafish brains were divided into 40,000 g supernatant and pellet fractions, and an Ettan 2-D fluorescence difference gel electrophoresis (DIGE) system was used to detect ethanol-induced alterations in the level of protein expression. Protein identification was carried out using matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and the Mascot and ProFound search engines. In the present study, we have identified some novel protein targets as well as substantiated some putative previous targets of chronic ethanol exposure.

Introduction

The central nervous system (CNS) appears to be an especially sensitive target for ethanol with chronic alcohol administration inducing adaptive changes that are manifested as tolerance and physical dependence as well as resulting in neurotoxicity. The molecular mechanisms responsible for these changes have not been fully elucidated, but most likely involve alterations in protein activity, level of expression, intracellular localization or post-translational modification. Genomic studies have identified changes in the expression of a number of genes belonging to diverse functional groups after chronic ethanol exposure (Liu et al., 2004, Mayfield et al., 2002). Most pronounced and consistent changes were obtained in gene families encoding mitochondrial proteins, proteins involved in signal transduction and synaptic transmission, as well as myelin-related proteins (Liu et al., 2004, Mayfield et al., 2002, Sokolov et al., 2003). Identification of ethanol-sensitive genes is important for a complete understanding of the molecular effects of ethanol. However, differences in mRNA expression are not reliable predictors of differences in protein expression (Chen et al., 2002, Greenbaum et al., 2003, Gygi et al., 1999). Alcohol administration has been shown to alter protein translation without changing mRNA levels (Dodd and Lewohl, 1998). In addition, genomic studies do not provide insight into post-translational modifications of proteins. Thus, a necessary step in understanding the effects of chronic ethanol exposure requires characterizing the ethanol-induced protein changes.

Because of recent advances in electrophoresis and mass spectrometry, a more global investigation of the effects of chronic ethanol treatment on brain proteins (i.e., the brain proteome) is now technologically possible. In the present study, the Ettan 2-D fluorescence difference gel electrophoresis (DIGE) system and MALDI-TOF mass spectrometry were used to identify proteins whose level of expression was altered by chronic ethanol exposure. By incorporating the use of an internal standard and by allowing controls and experimental groups to be run on the same gel, the Ettan DIGE system reduces intergel variability and simplifies protein spot matching thus facilitating quantification of protein differences. Current studies were carried out using the long-fin striped strain of zebrafish Danio rerio. Zebrafish offer a number of benefits for studying the CNS effects of ethanol (Dlugos and Rabin, 2003, Gerlai et al., 2000). Besides advantages in cost and ease of breeding and maintenance, zebrafish are readily amenable to genetic manipulation. In addition, the DNA sequences of humans and zebrafish are similar (Barbazuk et al., 2000). Furthermore, zebrafish are sensitive to pharmacological concentrations of ethanol and exhibit strain differences in terms of sensitivity and tolerance to ethanol (Dlugos and Rabin, 2003).