Early-Life Neuronal-Specific Iron Deficiency Alters the Adult Mouse Hippocampal Transcriptome

Abstract

Background: Iron deficiency (ID) compromises the developing nervous system, including the hippocampus, resulting in later-life deficits despite iron repletion. The iron-dependent molecular changes driving these lasting deficits, and the effect of early iron repletion, are incompletely understood. Previous studies have utilized dietary models of maternal-fetal ID anemia (IDA) to address these questions; however, concurrent anemia prevents delineation of the specific role of iron.

Objective: The aim of the study was to isolate the effects of developmental ID on adult hippocampal gene expression and to determine if iron repletion reverses these effects in a mouse model of nonanemic hippocampal neuronal ID.

Methods: Nonanemic, hippocampus-specific neuronal ID was generated by using a Tet-OFF dominant negative transferrin receptor (DN-TFR1) mouse model that impairs cellular iron uptake. Hippocampal ID was reversed with doxycycline at postnatal day 21 (P21) in a subset of mice to create 2 experimental groups, chronically iron-deficient and formerly iron-deficient mice, which were compared with their respective doxycycline-treated and untreated iron-sufficient controls. RNA from adult male hippocampi was sequenced. Paired-end reads were analyzed for differential expression. Differentially expressed genes were analyzed in Ingenuity Pathway Analysis.

Results: A total of 346 genes were differentially expressed in adult, chronically iron-deficient hippocampi compared with controls. ID dysregulated genes in critical neurodevelopmental pathways, including axonal guidance, CDK5, Ephrin receptor, Rac, and Neurotrophin/Trk signaling. Iron repletion at P21 normalized adult hippocampal expression of 198 genes; however, genes involved in cAMP response element-binding protein (CREB) signaling, neurocognition, and neurologic disease remained dysregulated in adulthood.

Conclusions: Chronic ID during development, independent of anemia, alters the adult mouse hippocampal transcriptome. Restoring iron status during a known critical period of hippocampal neurodevelopment incompletely normalized these changes, suggesting a need for additional studies to identify the most effective timeline for iron therapy, and adjunctive treatments that can fully restore ID-induced molecular changes, particularly in human populations in whom chronic ID is endemic.

FIGURE 1

Gene dysregulation in the CID and FID adult hippocampus. Chronic ID starting in late embryonic development results in dysregulation of 346 genes in the adult hippocampus. The majority of these genes are downregulated (P = 0.047). When chronic ID is reversed at P21, 148 genes are dysregulated in the adult hippocampus. The majority are upregulated (P < 0.0001). Of these 148 genes, 58 are genes that are also dysregulated in the CID hippocampus. The remaining 90 dysregulated genes are uniquely dysregulated in the FID hippocampus. CID, chronically iron deficient; FID, formerly iron deficient; ID, iron deficiency; IS, iron sufficient; P, postnatal day.

FIGURE 2

Critical neuronal signaling pathways are altered by ID and rescued by its reversal at P21. In IPA, differentially expressed genes were mapped to canonical signaling pathways. (A) In the adult CID hippocampus, dysregulated genes mapped significantly onto canonical signaling pathways that function in neurodevelopment and neuroplasticity [P < 0.05, −log(P value) > 1.3; dotted horizontal line]. On the basis of the genes that were dysregulated and the directionality of their dysregulation, it was predicted that the majority of these pathways would have a net decrease in activity, indicated by a negative z score (z <−1.5). (B) In the adult FID hippocampus, dysregulated genes no longer mapped significantly to the majority of these same pathways. Dysregulated genes still mapped significantly to axonal guidance signaling and CXCR4 signaling pathways; however, there was no predicted effect on the net function of the pathways. A list of definitions of gene and protein names used in this figure is included in Supplemental Materials. CID, chronically iron deficient; FID, formerly iron deficient; ID, iron deficiency; IPA, Ingenuity Pathway Analysis; IS, iron sufficient.

FIGURE 3

Genes permanently dysregulated by ID are involved in neurocognitive function and dysfunction. (A) Of the 58 genes dysregulated in both the CID and FID hippocampus, 9 (15%) are downregulated in both conditions and 30 (52%) are upregulated in both conditions. The remaining 19 genes (33%) are dysregulated with opposite directionality in the 2 conditions. The upward arrows indicate gene upregulation; downward arrows indicate gene downregulation. (B) When mapped to diseases and functions in IPA, these genes map to the functions of cognition and learning as well as schizophrenia and Alzheimer disease. A list of definitions of gene and protein names used in this figure is included in Supplemental Materials. CID, chronically iron deficient; FID, formerly iron deficient; ID, iron deficiency; IPA, Ingenuity Pathway Analysis; IS, iron sufficient.

FIGURE 4

CREB1 signaling is predicted to be altered by ID. (A) Thirteen genes involved in CREB1 signaling were found to be significantly downregulated in the CID hippocampus. On the basis of these gene expression changes, CREB1 activity is predicted by IPA to be significantly decreased (z score = −3.42, P < 0.001). (B) In the FID hippocampus, 7 CREB1-regulated genes were found to be significantly upregulated, which predicts a significant increase in CREB1 activity (z score = 2.61, P < 0.001). Genes outlined in bold are dysregulated in both CID and FID hippocampus. A list of definitions of gene and protein names used in this figure is included in Supplemental Materials. CID, chronically iron deficient; CREB1, cAMP response element-binding protein 1; FID, formerly iron deficient; ID, iron deficiency; IPA, Ingenuity Pathway Analysis.