Differential Early Mechanistic Frontal Lobe Responses to Choline Chloride and Soy Isoflavones in an Experimental Model of Fetal Alcohol Spectrum Disorder

Abstract

Fetal alcohol spectrum disorder (FASD) is the most common preventable cause of neurodevelopmental defects, and white matter is a major target of ethanol neurotoxicity. Therapeutic interventions with choline or dietary soy could potentially supplement public health preventive measures. However, since soy contains abundant choline, it would be important to know if its benefits are mediated by choline or isoflavones. We compared early mechanistic responses to choline and the Daidzein+Genistein (D+G) soy isoflavones in an FASD model using frontal lobe tissue to assess oligodendrocyte function and Akt-mTOR signaling. Long Evans rat pups were binge administered 2 g/Kg of ethanol or saline (control) on postnatal days P3 and P5. P7 frontal lobe slice cultures were treated with vehicle (Veh), Choline chloride (Chol; 75 µM), or D+G (1 µM each) for 72 h without further ethanol exposures. The expression levels of myelin oligodendrocyte proteins and stress-related molecules were measured by duplex enzyme-linked immunosorbent assays (ELISAs), and mTOR signaling proteins and phosphoproteins were assessed using 11-plex magnetic bead-based ELISAs. Ethanol's main short-term effects in Veh-treated cultures were to increase GFAP and relative PTEN phosphorylation and reduce Akt phosphorylation. Chol and D+G significantly modulated the expression of oligodendrocyte myelin proteins and mediators of insulin/IGF-1-Akt-mTOR signaling in both control and ethanol-exposed cultures. In general, the responses were more robust with D+G; the main exception was that RPS6 phosphorylation was significantly increased by Chol and not D+G. The findings suggest that dietary soy, with the benefits of providing complete nutrition together with Choline, could be used to help optimize neurodevelopment in humans at risk for FASD.

Keywords: FASD; choline; soy.

Figure 4

Ethanol and treatment (Chol and D+G) effects on intermediate signaling molecules in the Insulin/IGF-1-Akt-mTOR pathway. (A) Akt, (B) ^pS473-Akt, (C) p/T-Akt, (D) PTEN, (E) ^pS380-PTEN, (F) p/T-PTEN. Graphs depict the mean ± SD of (N = 6 cultures/group; FLU = fluorescent light units). Inter-group differences were analyzed by a two-way ANOVA (see Table 3) with post-hoc multiple comparisons using Tukey tests. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 1

Ethanol and treatment (Chol and D+G) effects on (A) MAG1, (B) MOG, (C) MBP, (D) PLP, (E) PDGFR-α, and (F) GALC in rat frontal lobe slice cultures. Immunoreactivity was measured by duplex ELISA, with results normalized to RPLPO. Graphs depict the mean ± SD of results (N = 6 cultures/group). Inter-group differences were analyzed by two-way ANOVA (see Table 1) with post-hoc multiple comparisons using Tukey tests. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 2

Ethanol and treatment (Chol and D+G) effects on (A) GFAP, (B) Ubiquitin, (C) 8-OHdG, and (D) HNE measured by duplex ELISA. Graphs depict the mean ± SD of immunoreactivity (N = 6 cultures/group). Inter-group differences were analyzed by two-way ANOVA (see Table 1) with post-hoc multiple comparisons using Tukey tests. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 3

Ethanol and treatment (Chol and D+G) effects on upstream Insulin/IGF-1-Akt-mTOR pathway molecules. (A) Insulin R, (B) ^{pYpY1162/1163}-Insulin-R, (C) p/T-Insulin-R, (D) IGF-1R, (E) ^{pYpY1135/1136}-IGF-1R, (F) p/T-IGF-1R, (G) IRS1, (H) ^pS636-IRS-1, and (I) p/T-IRS1. Graphs depict the mean ± SD of results (N = 6 cultures/group; FLU = fluorescent light units). Inter-group differences were analyzed by two-way ANOVA (see Table 2) with post-hoc multiple comparisons using Tukey tests. * p < 0.05; ** p < 0.01; **** p < 0.0001.

Figure 5

Ethanol and treatment (Chol and D+G) effects on intermediate key signaling molecules in the Insulin/IGF-1-Akt-mTOR pathway. (A) GSK-3α, (B) ^pS21-GSK-3α, (C) p/T-GSK-3α, (D) GSK-3β, (E) ^pS9-GSK-3β, and (F) p/T-GSK-3β. Graphs depict the mean ± SD of immunoreactivity (N = 6 cultures/group; FLU = fluorescent light units). Inter-group differences were analyzed by two-way ANOVA (see Table 3) with post-hoc Tukey tests. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 6

Ethanol and treatment (Chol and D+G) effects on the downstream signaling molecules in the Insulin/IGF-1-Akt-mTOR pathway. (A) TSC2, (B) ^pS939-TSC2, (C) p/T-TSC2, (D) mTOR, (E) ^pS2448-mTOR, (F) p/T-mTOR. Graphs depict the mean ± SD of immunoreactivity (N = 6 cultures/group; FLU = fluorescent light units). Inter-group differences were analyzed by a two-way ANOVA (see Table 4) with post-hoc multiple comparisons using Tukey tests. * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Figure 7

Ethanol and treatment (Chol and D+G) effects on key downstream signaling molecules in the Insulin/IGF-1-Akt-mTOR pathway. (A) p70S6K, (B) ^pT412-p70S6K, (C) p/T-p70S6K, (D) RPS6, (E) ^pS235/236-RPS6, (F) p/T-RPS6. Graphs depict the mean ± SD of results (N = 6 cultures/group; FLU = fluorescent light units). Inter-group differences were analyzed by a two-way ANOVA (see Table 4) with post-hoc multiple comparisons using Tukey tests. * p < 0.05; ** p < 0.01; *** p < 0.001.