Neuroprotective peptide ADNF-9 in fetal brain of C57BL/6 mice exposed prenatally to alcohol

Abstract

Background: A derived peptide from activity-dependent neurotrophic factor (ADNF-9) has been shown to be neuroprotective in the fetal alcohol exposure model. We investigated the neuroprotective effects of ADNF-9 against alcohol-induced apoptosis using TUNEL staining. We further characterize in this study the proteomic architecture underlying the role of ADNF-9 against ethanol teratogenesis during early fetal brain development using liquid chromatography in conjunction with tandem mass spectrometry (LC-MS/MS).

Methods: Pregnant C57BL/6 mice were exposed from embryonic days 7-13 (E7-E13) to a 25% ethanol-derived calorie [25% EDC, Alcohol (ALC)] diet, a 25% EDC diet simultaneously administered i.p. ADNF-9 (ALC/ADNF-9), or a pair-fed (PF) liquid diet. At E13, fetal brains were collected from 5 dams from each group, weighed, and frozen for LC-MS/MS procedure. Other fetal brains were fixed for TUNEL staining.

Results: Administration of ADNF-9 prevented alcohol-induced reduction in fetal brain weight and alcohol-induced increases in cell death. Moreover, individual fetal brains were analyzed by LC-MS/MS. Statistical differences in the amounts of proteins between the ALC and ALC/ADNF-9 groups resulted in a distinct data-clustering. Significant upregulation of several important proteins involved in brain development were found in the ALC/ADNF-9 group as compared to the ALC group.

Conclusion: These findings provide information on potential mechanisms underlying the neuroprotective effects of ADNF-9 in the fetal alcohol exposure model.

Figure 1

Neuroprotective effect of ADNF-9 in fetal brains exposed prenatally to alcohol at E13. Prenatal alcohol exposure induced significant reduction in fetal brain weight in the ALC group as compared to the PF group (p < 0.01). ADNF-9 administration alongside prenatal alcohol exposure prevented alcohol-induced reduction in fetal brains weights (p < 0.05). Values are expressed as means ± SEM. N = 5 for each group. *p < 0.05, **p < 0.01 (Newman-Keul's post hoc test).

Figure 2

Neuroprotective effect of ADNF-9 against alcohol-induced cell death in primordium cingulate cortex at E13. Prenatal alcohol exposure induced increases in TUNEL-positive cells. Importantly, administration of ADNF-9 prevented the alcohol-induced increases in cell death (a-c). Note that cells undergoing apoptosis are indicated by cell processes as shown by arrowheads. However, arrows indicate cells in the final stage of apoptosis. Statistical analyses demonstrate a significant difference between groups (p = 0.0405). (d) Prenatal alcohol exposure induced significant increases in the number of TUNEL-positive cells in the ALC group as compared to the PF (p < 0.05). ADNF-9 administration prevented significantly the alcohol-induced increases in the number of TUNEL-positive cells (p < 0.05). Values are expressed as means ± SEM. N = 4 for each group. *p < 0.05 (Newman-Keul's post hoc test). Scale bar = 100 μm.

Figure 3

PCA score plot of the levels of the identified proteins for the analyzed groups: ALC and ALC/SAL(ADNF-9).

Figure 4

Proteins that are significantly upregulated in the ALC/ADNF-9 group as compared to the ALC group, cyclin-dependent kinase inhibitor 1B (a), and nuclear cap-binding protein subunit 1 (b).