Iron is essential for neuron development and memory function in mouse hippocampus

Abstract

Iron deficiency (ID) is the most prevalent micronutrient deficiency in the world and it affects neurobehavioral outcome. It is unclear whether the effect of dietary ID on the brain is due to the lack of neuronal iron or from other processes occurring in conjunction with ID (e.g. hypoxia due to anemia). We delineated the role of murine Slc11a2 [divalent metal ion transporter-1 (DMT-1)] in hippocampal neuronal iron uptake during development and memory formation. Camk2a gene promoter-driven cre recombinase (Cre) transgene (Camk2a-Cre) mice were mated with Slc11a2 flox/flox mice to obtain nonanemic Slc11a2(hipp/hipp) (double mutant, hippocampal neuron-specific knockout of Slc11a2(hipp/hipp)) mice, the first conditionally targeted model of iron uptake in the brain. Slc11a2(hipp/hipp) mice had lower hippocampal iron content; altered developmental expression of genes involved in iron homeostasis, energy metabolism, and dendrite morphogenesis; reductions in markers for energy metabolism and glutamatergic neurotransmission on magnetic resonance spectroscopy; and altered pyramidal neuron dendrite morphology in area 1 of Ammon's Horn in the hippocampus. Slc11a2(hipp/hipp) mice did not reach the criterion on a difficult spatial navigation test but were able to learn a spatial navigation task on an easier version of the Morris water maze (MWM). Learning of the visual cued task did not differ between the Slc11a2(WT/WT) and Slc11a2(hipp/hipp) mice. Slc11a2(WT/WT) mice had upregulation of genes involved in iron uptake and metabolism in response to MWM training, and Slc11a2(hipp/hipp) mice had differential expression of these genes compared with Slc11a2(WT/WT) mice. Neuronal iron uptake by DMT-1 is essential for normal hippocampal neuronal development and Slc11a2 expression is induced by spatial memory training. Deletion of Slc11a2 disrupts hippocampal neuronal development and spatial memory behavior.

Figures

FIGURE 1

Regional iron concentrations in brains of Slc11a2WT/WT and Slc11a2hipp/hipp mice. Values are means ± SEM [Slc11a2WT/WT: n = 9 total brains, 14 cortices, 13 cerebellum pools (4 cerebella/pool), 6 hippocampus pools (14 hippocampi/pool); Slc11a2hipp/hipp: n = 4 total brains, 11 cortices, 7 cerebellum pools (4 cerebella/pool), 4 hippocampus pools (8 hippocampi/pool)]. *Different from Slc11a2hipp/hipp, P < 0.05.

FIGURE 2

Perl's staining in hippocampus at P25 in Slc11a2WT/WT (n = 4) (A,C) and Slc11a2hipp/hipp (B,D) (n = 4) mice. Photomicrographs are at 100× (A,B) and 200× (C,D). Hippocampal area CA1 pyramidal neuronal soma in Slc11a2hipp/hipp mice show less staining (arrows) than Slc11a2WT/WT mice (arrowheads), whereas nonpyramidal cells localized between the hippocampal CA subfields and dentate gyrus retain positivity.

FIGURE 3

Hippocampal gene expression of Slc11a2 exon 7 (A) and TfRc (B) in Slc11a2WT/WT and Slc11a2hipp/hipp mice from P5 to P90. Values are means ± SEM, n = 3–5. Significant effects (P < 0.05): A, Cre status, Age, Cre status × Age ; B, Cre status, Age.

FIGURE 4

Hippocampus metabolite concentrations of Slc11a2hipp/hipp and Slc11a2WT/WT mice at P90. Values are means ± SEM, n = 6. *Different from Slc11a2hipp/hipp, P < 0.05. Lac, lactate; PCr, phosphocreatine.

FIGURE 5

Visualization of pyramidal apical dendrite main shaft lengths in area CA1 of hippocampus of P45 Slc11a2WT/WT (n = 9) (A,C) and Slc11a2hipp/hipp (n = 4) (B,D) mice. Cell bodies are oriented longitudinally across the upper right of each panel, directionally denoted by the arrow. Apical dendrites in Slc11a2WT/WT mice and Slc11a2hipp/hipp mice are marked with arrowheads. Magnification at × 200 (A,B) and × 400 (C,D).

FIGURE 6

Spatial navigation memory in 3-mo-old Slc11a2hipp/hipp (n = 15) and Slc11a2WT/WT (n = 12) mice in the standard MWM (version 1). Values are means ± SEM. (A) Percent of total swim time spent in target quadrant on probe trials after each day of training. Significant effects (P < 0.05): Cre status, Training, Cre status × Training. (B) Percent time spent floating during probe trials. Significant effects (P < 0.05): Cre status, Training. (C) Mean escape latencies by trial across 3 training days. Significant effects (P < 0.05): Cre status, Training. (D) Percent time spent in target quadrant by trial across 3 training days. Significant effects (P < 0.05): Cre status, Training, Cre status × Training. (E) Mean swim velocity by trial across 3 training days. Significant effects (P < 0.05): Cre status, Training, Cre status × Training

FIGURE 7

Gene expression in untrained, trained on hard MWM version 1, and trained on easier MWM version 2 Slc11a2WT/WT and Slc11a2hipp/hipp mice on P 90, quantified by qPCR. In each panel, the Y-axis represents units of relative mRNA expression with a value of 1.0 set for untrained Slc11a2WT/WT mice. Values are means ± SEM, n = 7–10 or 4 (untrained). Asterisks indicate that the designated groups differ: *P < 0.05; **P < 0.01 (unpaired 2-tailed Student's t tests). Significant effects in the 2-way ANOVA were: (A) Slc11a2; Cre status, MWM version, Cre status × MWM version; (B) Tfrc; Cre status, MWM version; (C) Aco1; MWM version; (D) Ireb2; Cre status, MWM version; (E) Grin2b; Cre status × MWM version; (F) Camk2a; MWM version, Cre status × MWM version.