Prenatal Choline Supplementation Diminishes Early-Life Iron Deficiency-Induced Reprogramming of Molecular Networks Associated with Behavioral Abnormalities in the Adult Rat Hippocampus

Abstract

Background: Early-life iron deficiency is a common nutrient deficiency worldwide. Maternal iron deficiency increases the risk of schizophrenia and autism in the offspring. Postnatal iron deficiency in young children results in cognitive and socioemotional abnormalities in adulthood despite iron treatment. The rat model of diet-induced fetal-neonatal iron deficiency recapitulates the observed neurobehavioral deficits.

Objectives: We sought to establish molecular underpinnings for the persistent psychopathologic effects of early-life iron deficiency by determining whether it permanently reprograms the hippocampal transcriptome. We also assessed the effects of maternal dietary choline supplementation on the offspring's hippocampal transcriptome to identify pathways through which choline mitigates the emergence of long-term cognitive deficits.

Methods: Male rat pups were made iron deficient (ID) by providing pregnant and nursing dams an ID diet (4 g Fe/kg) from gestational day (G) 2 through postnatal day (PND) 7 and an iron-sufficient (IS) diet (200 g Fe/kg) thereafter. Control pups were provided IS diet throughout. Choline (5 g/kg) was given to half the pregnant dams in each group from G11 to G18. PND65 hippocampal transcriptomes were assayed by next generation sequencing (NGS) and analyzed with the use of knowledge-based Ingenuity Pathway Analysis. Real-time polymerase chain reaction was performed to validate a subset of altered genes.

Results: Formerly ID rats had altered hippocampal expression of 619 from >10,000 gene loci sequenced by NGS, many of which map onto molecular networks implicated in psychological disorders, including anxiety, autism, and schizophrenia. There were significant interactions between iron status and prenatal choline treatment in influencing gene expression. Choline supplementation reduced the effects of iron deficiency, including those on gene networks associated with autism and schizophrenia.

Conclusions: Fetal-neonatal iron deficiency reprograms molecular networks associated with the pathogenesis of neurologic and psychological disorders in adult rats. The positive response to prenatal choline represents a potential adjunctive therapeutic supplement to the high-risk group.

Keywords: choline supplementation; fetal iron deficiency; hippocampus; psychological disorders; transcriptome.

FIGURE 1

RNA-Seq data generated from PND65 male rat hippocampus following gestational iron deficiency with or without choline supplementation. (A) Flow chart of RNA sample preparation and sequencing by Illumina HiSeq. The right panel shows total NGS reads from RNA libraries for each diet group. (B) Log(FC) plot shows differentially expressed genes between FID and IS data sets. Ave., average; bp, base pairs; CPM, count per million; Down, downregulated. FC, fold change; FID, formerly iron-deficient; FID-C, FID choline; IS, iron sufficient; IS-C, IS choline; NGS, next generation sequencing; nt, nucleotides; PND, postnatal day; Up, upregulated.

FIGURE 2

Annotated functions associated with dysregulated genes in the hippocampus on PND65 of FID rats. A number of downregulated factors (A) are known to be involved in neurotransmission and long-term potentiation, whereas a greater number of upregulated factors (B) are implicated in the pathogenesis of Alzheimer disease, autism, schizophrenia, and anxiety disorders. FID, formerly iron deficient; PND, postnatal day. A list of definitions of gene and protein names used in this figure is included in Supplemental Material.

FIGURE 3

Altered mTOR/p70S6K (A) and dopamine/BDNF/CDK5 (B) signaling pathways in the hippocampus on PND65 of FID rats. BDNF, brain-derived neurotrophic factor; CDK5, cyclin-dependent kinase 5; FID, formerly iron deficient; mTOR, mechanistic target of rapamycin; PND, postnatal day. A list of definitions of gene and protein names used in this figure is included in Supplemental Material.

FIGURE 4

Prenatal choline supplementation partially normalized dysregulated genes in the hippocampus on PND65 of FID rats. (A) Scatter plot showing differentially expressed genes between the FID-C and FID groups. (B) Heat map showing gene clusters from all treatment groups. The key indicates differential gene expression by diet. Overlapped regions of Venn diagrams depict genes (127 downregulated and 181 upregulated) altered in the FID and normalized in the FID-C group. (C) Interactive gene networks implicated in the pathogenesis of Alzheimer disease, schizophrenia, autism, and mood disorders that were normalized by prenatal choline treatment of the FID group. CPM, counts per million; Down, downregulated; FC, fold change; FID, formerly iron deficient; FID-C, FID-choline; IS, iron sufficient; IS-C, IS choline; PND, postnatal day; Up, upregulated. A list of definitions of gene and protein names used in this figure is included in Supplemental Material.

FIGURE 5

Prenatal choline supplementation reprogrammed the hippocampal transcriptome of PND65 IS-C rats. (A) Bar graphs showing functions that are associated with dysregulated genes in the transcriptome of IS-C rats. The key above the graph explains the number of altered genes and direction of change relative to IS rats. (B) Gene networks altered in the IS-C hippocampus that are implicated in the cognitive impairment and the pathogenesis of schizophrenia, major depression, and Alzheimer disease. Down, downregulated; IS, iron sufficient; IS-C, IS choline; PND, postnatal day; Up, upregulated. A list of definitions of gene and protein names used in this figure is included in Supplemental Material.