Fetal regional brain protein signature in FASD rat model

Abstract

Fetal alcohol spectrum disorders (FASD) describe neurodevelopmental deficits in children exposed to alcohol in utero. We hypothesized that gestational alcohol significantly alters fetal brain regional protein signature. Pregnant rats were binge-treated with alcohol or pair-fed and nutritionally-controlled. Mass spectrometry identified 1806, 2077, and 1456 quantifiable proteins in the fetal hippocampus, cortex, and cerebellum, respectively. A stronger effect of alcohol exposure on the hippocampal proteome was noted: over 600 hippocampal proteins were significantly (P < .05) altered, including annexin A2, nucleobindin-1, and glypican-4, regulators of cellular growth and developmental morphogenesis. In the cerebellum, cadherin-13, reticulocalbin-2, and ankyrin-2 (axonal growth regulators) were significantly (P < .05) altered; altered cortical proteins were involved in autophagy (endophilin-B1, synaptotagmin-1). Ingenuity analysis identified proteins involved in protein homeostasis, oxidative stress, mitochondrial dysfunction, and mTOR as major pathways in the cortex and hippocampus significantly (P < .05) affected by alcohol. Thus, neurodevelopmental protein changes may directly relate to FASD neuropathology.

Keywords: Alcohol; FASD; Fetal; Teratology.

Figure 1. Proteomic changes in the fetal hippocampus following prenatal alcohol exposure

(A) Volcano plot of protein abundance and P-values (n = 16 per group) following gestational alcohol exposure, shows the distribution of proteins (T-test P value (log₂)) versus the abundance difference (log₂ (EtOH/Pair-fed control)). Colored dots represent significantly (P < 0.05) upregulated (red) or downregulated (blue) proteins, black lines represent the significance thresholds post multiple comparison correction (Permutation testing; P < 0.05); (B) Functional enrichment analysis of significantly upregulated proteins; (C) INGENUITY pathways analysis (IPA) of canonical pathways altered following gestational alcohol exposure; and (D) IPA of toxicity functions altered following gestational alcohol exposure.

Figure 2. Proteomic changes in the cortex following prenatal alcohol exposure

(A) Volcano plot of cortical proteins abundance and P-values (n = 16 per group) following gestational alcohol exposure. (B) INGENUITY pathway analysis (IPA) of canonical pathways of significantly altered proteins. (C) IPA of toxicity of significantly altered proteins.

Figure 3. Proteomic changes in the cerebellum following prenatal alcohol exposure

(A) Volcano plot of cerebellar proteins abundance and P-values (n = 16 per group) following gestational alcohol exposure; and (B) INGENUITY pathway analysis (IPA) of canonical pathways altered following gestational alcohol exposure.

Figure 4. Venn diagram comparing the number of proteins which are significantly dysregulated in abundance following alcohol-exposed compared to pair-fed control rats across the three brain proteomic analyses

The protein “ras-related protein Rab-21” was found to be dysregulated in all three fetal brain regions after gestational alcohol exposure. Overlapping regions indicate the number of shared significantly altered proteins (EtOH/Pair-fed control) between the respective brain regions.