Differential expression of proteins in fetal brains of alcohol-treated prenatally C57BL/6 mice: a proteomic investigation

Abstract

Alcohol is known to impede the growth of the central nervous system and to induce neurodegeneration through cellular apoptosis. We have previously shown that moderate prenatal alcohol exposure results in brain defects at different stages of development. In this study, we further characterize the proteomic architecture underlying ethanol teratogenesis during early fetal brain development using chromatography in conjunction with a LC-MS/MS system. Pregnant C57BL/6 mice were exposed from embryonic day 7 (E7) to E13 with either a 25% ethanol derived calorie or pair-fed liquid diets. At E13, fetal brains were collected from five dams for each group. Individual brains were homogenized and the extracted proteins were then tryptically digested and analyzed by LC-MS/MS. Label-free quantitative proteomic analyses were performed on proteomes extracted from fetal brains of both alcohol-treated (ALC) and pair-fed groups. These analyses demonstrated that prenatal alcohol exposure induced significant downregulation (p<0.001) of the expression of mitochondrial enzymes including ADP/ATP translocase 1, ATP synthase subunit alpha and ubiquinol-cytochrome-c reductases. In addition, mitochondrial carrier homolog 1, which plays a role in apoptosis, was significantly downregulated (p<0.001) in the ALC group. Moreover, among the cytosolic proteins that were significantly downregulated (p<0.001) are Bcl-2, 14-3-3 protein and calmodulin. Significant downregulation (p<0.001) of proteins that are critical for fetal brain development was observed such as prohibitin and neuronal migration protein doublecortin. These findings provide information about possible mechanisms underlying the effects of prenatal alcohol exposure during early embryonic stage.

Figure 1

Prenatal alcohol exposure increases cell death in primordium frontal cortex (FCx) and basal ganglia eminence (BGE) (b) as compared to PF group (a) at E13 as indicated by TUNEL staining. High power photomicrographs show sections of FCx that are pointed by arrows in PF (c) and ALC (d) groups. Arrowheads indicate cell undergoing apoptosis. Scale bars: 100 μm.

Figure 2

Classification of identified proteins expressed as total percentage (a). Majority of identified proteins are enzymes (26%), nucleotide binding proteins (18%) and other isoforms. Further classification of the enzyme subgroup includes 17% kinase, 14% dehydrogenase, 13% isomerase, and 12% cytochrome c oxidase (b).

Figure 3

PCA score plot of the areas of the identified proteins for the two groups analyzed: control group (Pairfed, PF) and alcohol-treated group (ALC). Insets are enlarged regions of the plot.

Figure 4

Dot-plots of the areas of some mitochondrial proteins that have demonstrated statistical differences between the two groups, ADP/ATP translocase (a), ATP synthase α-subunit (b), prohibitin (c), and ubiquinol-cytochrome-c reductase complex core protein I (d).

Figure 5

Dot-plots of the areas of some nuclear proteins that have demonstrated statistical differences between the two groups, heterogeneous nuclear ribonucleoprotein H (a), mediator of DNA damage checkpoint protein 1 (b), and probable ATP-dependent RNA helicase (c).

Figure 6

Scatter plots of protein intensities between two individual fetal brains from the same litter in the PF group (a, b) and ALC group (c, d). Significant correlation of protein intensities was found between two individual fetal brains in both PF and ALC group.