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Fetal-neonatal iron deficiency acutely alters hippocampal biochemistry, morphology, neurotransmission and electrophysiology resulting in short-term behavioral impairments. It also down-regulates brain-derived neurotrophic factor (BDNF) expression accompanied by the up-regulation of nerve growth factor (NGF), epidermal growth factor (EGF), glial-derived neurotrophic factor (GDNF), and p75 neurotrophic receptor. However, the etiology of long-term hippocampal neural transmission abnormalities and learning impairments remains unclear. Since BDNF modulates learning and memory, we assessed its expression in formerly iron deficient (FID) adult rats that had been iron deficient during the fetal and neonatal periods. BDNF was down-regulated in FID rats, whereas NGF, EGF and GDNF levels were similar to the always iron sufficient control group. Consistent with attenuated BDNF activity, we found lower expression of transcriptional targets of BDNF signaling in FID rats, including activity-dependent immediate early genes (e.g., c-fos, early-growth response gene-1 and -2) and the rate-limiting enzyme of cholesterol synthesis 3-hydroxy-3-methylglutaryl coenzyme A reductase. Our findings show that fetal-neonatal iron deficiency lowers BDNF function beyond the period of iron deficiency in the hippocampus. The lower adult hippocampal BDNF activity may underlie the persistence of learning deficits seen after early-life iron deficiency.
Iron deficiency is a common early-life nutrient deficiency affecting approximately 30-50% pregnancies worldwide, including an estimated 80% of pregnancies in developing countries (1). Late gestational and neonatal iron deficiency arises from four maternal conditions during pregnancy: severe iron deficiency anemia, placental vascular insufficiency resulting from maternal hypertension, diabetes mellitus and cigarette smoking (2-5). In humans, neonatal iron deficiency causes deficits in cognitive function during the period of iron deficiency, and poor school performance beyond the period of iron deficiency (6, 7). With early iron deficiency, although certain developmental deficits can be corrected with iron treatment, other behavioral and cognitive deficits persist beyond 10 years after iron treatment (8). The neural basis of these long-term deficits remains unclear. However, evidence from animal models suggest that fetal-neonatal iron deficiency influences multiple neuronal processes such as myelination, monoamine metabolism and energy metabolism (9-13).
Acutely, fetal-neonatal brain iron deficiency affects hippocampal development and function as evidenced by decreased energy metabolism, impaired neuronal morphology and transmission, and increased susceptibility to infarction in the neonatal period (12-15). Our group has described the specific effects of fetal-neonatal iron deficiency on the expression of neurotrophic factors critical for inducing and maintaining hippocampal differentiation and plasticity (16). Hippocampal brain-derived neurotrophic factor (BDNF) expression is down-regulated while nerve growth factor (NGF), epidermal growth factor (EGF), and glial-derived neurotrophic factor (GDNF) are upregulated during iron deficiency in rats (16). BDNF regulates multiple aspects of hippocampal development and function (17-19). In particular, induction of long-term potentiation (LTP), a cellular phenomenon associated with memory formation, in the rodent hippocampus rapidly increases BDNF transcript levels (20-22). Suppression of BDNF expression and genetic-deletion of BDNF lead to impairment of learning and memory (23, 24). BDNF signaling is mediated by tyrosine-receptor kinase B (TrkB) and p75 neurotrophic receptor (p75NTR) (25). BDNF binding of TrkB promotes neurite outgrowth and synaptic plasticity in part through regulation of activity-dependent immediate early genes c-fos, early-growth-response-gene 1 and 2 (Egr-1 and Egr-2). In contrast, BDNF binding of p75NTR facilitates long-term depression and reduces neurite outgrowth (26-28).
While it is not surprising that multiple systems are disrupted while the brain is iron deficient, the underlying mechanisms for continued compromised function long after iron repletion remains unclear. Following iron-repletion, formerly iron deficient (FID) adult rats continue to demonstrate deficits on hippocampal-dependent tasks consistent with similar findings in humans (29, 30). The behavioral deficits in these rats corroborated the finding of lower LTP expression in a similar model (14). Given its role in modulating synaptic plasticity, we postulated lower BDNF activity in the hippocampus of FID rats. Here we present evidence that fetal-neonatal iron deficiency continues to down-regulate BDNF expression and BDNF activity beyond the period of iron deficiency, suggesting a long-term alteration in the programming of BDNF expression. The lower BDNF activity may be an important molecular underpinning for the persistent cognitive deficits in FID rats.
Timed-pregnant Sprague-Dawley rats were purchased from Harlan (Indianapolis, IN). Fetal-neonatal iron deficiency was induced as previously described in order to achieve a 40% loss of total brain iron at postnatal day (P)10 (11), a degree of brain iron deficiency equivalent to that seen in newborn humans (3). In this model, the hippocampus remains iron-deficient (ID) through P30 (25% loss) and is iron-sufficient (IS) by P56 (14). In brief, pregnant dams were maintained on an ID diet from gestational day 2 to P7, after which time the nursing dams were given the nonpurified IS control diet. Pups from dams given IS diet throughout the experiment served as IS controls. Litters were culled to 8 pups/litter. All pups were weaned at P21 and fed IS diet for the duration of the experiment. This study design has been previously utilized and lowers the hematocrit to 19% in the ID group (31). It reduces total brain iron concentration by 45% and hippocampal iron concentration by 60% in the ID pups compared to IS controls at P15 with complete recovery of hippocampal iron concentration by P65 (12, 14, 31). All animal experiments were carried out with the approval of the University of Minnesota Institutional Animal Care and Use Committee.
IS control (198 mg/kg iron, Rx 241632) and ID (3 mg/kg iron, Rx 247497) nonpurified diets were purchased from Harlan Teklad (Madison, WI). The compositions of both IS and ID diets have been described previously (11).
P65 male rats were killed by an intraperitoneal (i.p.) injection of Beuthanasia (100 mg/kg). Brains were removed and bisected along the midline. Hippocampus was dissected, flash-frozen in liquid Nitrogen, and stored at −80°C.
Brain iron concentrations were measured as previously described (14). In brief, P65 rats were deeply anesthetized with an i.p. injection of Beuthanasia (100 mg/Kg) and perfused transcardially with 0.9% NaCl. Immediately following brain removal from the skull, the hippocampus was dissected and rinsed thoroughly in saline solution. Hippocampal tissue was measured for wet weight and subsequently lyophilized for 72h. Lyophilized tissue was digested with 4:1 nitric/perchloric acid solution for 5d. Iron concentration was determined by atomic absorption spectroscopy. Values were compared to a standard curve generated from diluted stock iron standards (Sigma).
Total RNA was isolated from dissected hippocampus using an RNA-isolation kit (Stratagene, La Jolla, CA) and concentrations were measured by absorbance at 260 nm (A260/280) using a NanoDrop ND-1000 (NanoDrop Technologies, Inc., Wilmington, DE). Approximately 4 μg of total RNA was used to generate cDNA by reverse transcription using SuperScript III (Invitrogen, Carlsbad, CA) and random hexamer primers per manufacturer recommendation. The resulting cDNA was diluted 7-fold to give a final volume of 140 μL. All qPCR experiments were performed with 1/2 the manufacturer's recommended volume (Applied Biosystems Inc., Foster City, CA) consisting of 4 μl of diluted cDNA, 5μl 2X Taqman® qPCR Universal Mix (No AmpErase), and 0.5 μl 20X Taqman® Gene Expression Assay primer/probe mix. Thermocyling was carried out according to the manufacturer's protocol (ABI) using a MX3000P instrument (Stratagene, La Jolla, CA). Information about the qPCR probes/primers is listed in Table 1.
Protein isolation was carried out as described previously (32). In brief, flash-frozen hippocampal tissues were lysed by sonication. Approximately 31 μg of total protein was loaded and separated in 12% and 4-12% gradient SDS-PAGE gels (Invitrogen, Carlsbad, CA). Protein was transferred onto Nitrocellulose membranes (Pierce, Rockford, IL) using wet transfer (Invitrogen X-cell II Blot Module). Membranes were blocked in Blocking Buffer for Near Infrared Fluorescent Western Blotting (Rockland, Gilbertsville, PA) for 1h at room temperature. Membranes were incubated in primary antibody diluted in Blocking Buffer overnight at 4°C with rocking and rinsed in PBS with 0.1% Tween-20 (4X) to remove excess antibody. Membranes were incubated in secondary antibody diluted in Blocking Buffer with 0.1% Tween-20 and 0.01% SDS at room temperature for 45min, and rinsed 4X in PBS with 0.1% Tween-20 to remove excess antibody. For quantification, membranes were imaged with Odyssey Infrared Imaging System (Li-cor Biosciences, Lincoln, NE) and the integrated intensity of the protein of interest was standardized to actin whose expression is not affected by iron deficiency and acts as an internal control. The primary antibodies included anti-BDNF (1:1000) rabbit polyclonal (Abcam, Cambridge, MA), anti-TrkB (1:1000) rabbit monoclonal (Cell Signaling, Danvers, MA), and anti-actin (1:5000) mouse monoclonal (Sigma, St. Louis, MO). Secondary antibodies included Alexa Fluor® 680 conjugated anti-mouse (1:12500) (Invitrogen, Carlsbad, CA) and Infrared Dye 800 conjugated anti-rabbit antibody (1:12500) (Rockland, Gilbertsville, PA).
Data for transcript and protein levels were collected from six male rats for each dietary group. Data were analyzed by a non-parametric Mann-Whitney U-test. Significance was set at an alpha of 0.05. Graphs and statistical calculations were performed with GraphPad Prism (GraphPad Software Inc., San Diego, CA).
At P65, the mean hippocampal iron concentration of the FID group was not different from the always IS control group (IS: 6.79 ± 0.78 μg Fe/g wet weight, n = 7; FID: 6.66 ± 0.84 μg Fe/g wet weight, n = 7; P = 1.00).
P65 FID rats had lower BDNF-III and -IV mRNA expression as well as lower protein levels compared to control IS rats (Figure 1A-B). The expression of BDNF receptors, TrkB and p75NTR, was also assessed. The FID group had lower TrkB mRNA (total and long-form) but protein levels were similar to IS control levels (Figure 1C-D). p75NTR mRNA expression was not different between groups (Table 2). The mRNAs of ciliary neurotrophic factor, EGF, GDNF, and NGF were also not different between control and FID P65 rats (Table 2).
To assess whether lower BDNF expression in the FID P65 hippocampus leads to a decrease in BDNF activity, mRNA transcript levels of several known transcriptional targets of BDNF signaling, including the activity-dependent immediate early genes c-fos, Egr-1 and Egr-2, and the 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) (33-35), were examined. Consistent with lowered BDNF activity, the expression of these target genes was down-regulated in P65 FID rats (Figure 2).
We and others have established behavioral and cognitive deficits associated with fetal-neonatal iron deficiency anemia, many of which persist beyond the period of iron deficiency (1, 8). In particular, fetal-neonatal iron deficiency affects the hippocampal-dependent learning and memory formation in adult FID rats (29). This study demonstrates that fetal-neonatal iron deficiency results in long-term decreased BDNF activity without a compensatory increase in TrkB expression in the rat hippocampus. It also suggests a potential role of iron homeostasis in long-term programming of hippocampal BDNF expression. Given the central role of BDNF in modulating learning and memory function, the findings provide a possible new molecular basis for the persistent long-term cognitive deficits observed in fetal-neonatal iron deficiency.
Our previous study revealed lower mRNA and protein levels of calmodulin regulated kinase-IIα (CamKIIα) and post-synaptic density 95 (PSD95), critical factors for synaptic structure and plasticity in the hippocampus of FID P65 rats (32). We speculate that decreased BDNF contributes to the reduced expression of CamKIIα and PSD95, ultimately leading to the documented impairment of synaptic plasticity in the FID rat (14, 29, 36, 37). The translation of these proteins at the synapse is known to depend on BDNF and the mTOR intracellular signaling pathway (38, 39), both of which are compromised during iron deficiency (16, 32). mTOR integrates stimuli including growth factors and nutrient and energy availability to regulate protein translation and cellular growth (40).
C-fos, Egr-1, and Egr-2 are activity-dependent immediate early transcription factors that are regulated by BDNF administration (41), N-methyl-D-aspartate (NMDA) receptor activation (42), or exposure to an enriched environment (43, 44). These manipulations increase synaptic plasticity and LTP in the hippocampus. Thus, lower expression of c-fos, Egr-1 and Egr-2 suggests a loss of plasticity in the hippocampus of FID adult rats. This less plastic hippocampus could be realized at the structural level by altered dendritic morphology (11) accompanied by altered expression of molecules involved in guiding and mediating structural changes in response to experience (i.e., synaptic input from CA3) including PSD95, CamKIIα and chemokine-(C-X-C motif)-ligand-12 (32). Furthermore, HMGCR and synaptobrevin I (Vamp1) are important factors for the formation and release of synaptic vesicles and contribute to the fluidity of synaptic efficacy in response to experience (35, 45). Lower HMGCR and Vamp1 (32) expression in FID hippocampus further support a reduction of synaptic plasticity, and are consistent with reductions in paired-pulse facilitation, a measure of presynaptic plasticity, reported in this model (14). Although HMGCR is expressed in both neurons and astrocytes (35), BDNF regulates HMGCR expression specifically in neurons (35). We speculate that the association of reduced expression of BDNF and HMGCR in FID P65 hippocampus found in this study provides evidence that the long-term HMGCR effects are neuronal in origin and thus may be responsible for pyramidal cell dysfunction.
The long-term down-regulation of BDNF without compensatory TrkB expression suggests an effect of early-life iron deficiency on the dampening of this neurotrophic system potentially throughout the life span. Fundamentally, long-term abnormalities in hippocampal function following the abatement of iron deficiency can be ascribed to permanent changes in structure induced by the lack of iron during critical periods of development or to long-term dysregulation of genes important for optimal function. These are not mutually exclusive conditions. We have previously demonstrated the former possibility as evidenced by persistently abnormal dendritic structure at P65 in this model (12). The current study provides evidence of the latter possibility and is intriguing because this dysregulation exists in spite of re-establishment of nutritional adequacy. The mechanism of the effect is unknown but may relate to those involved in the developmental origins of health and disease (46), including epigenetic modifications (e.g., methylation and acetylation) of genes. The finding emphasizes the concept that provision of nutrients alone is not adequate to maintain optimal brain function, but that regulation by growth factors that ensure proper utilization of those nutrients is essential. Long-term dysregulation of these growth factors may thus account for persistent abnormal function in spite of nutrient repletion. These findings may also be an important part of understanding the early antecedents of adult neurological disorders characterized by reduced hippocampal function or early hippocampal degeneration, including Alzheimer and Parkinson diseases. Both are characterized by reduction of BDNF levels without compensatory increases in TrkB expression (19, 47). The altered expression of genes involved in the pathogenesis of Alzheimer disease (29) supports this possibility.
In summary, previous studies demonstrate the persistence of cognitive deficits in perinatal iron deficient children lasting beyond the period of iron treatment. Such deficits also occur in the rat model of perinatal iron deficiency. This animal model allowed the investigation into the molecular basis underlying these long-term deficits in the hippocampus, an area of the brain that subserves recognition memory. Our findings here corroborate the lower LTP expression and provide the basis for lower expression of factors (CamKIIα and PSD95) critical for synaptic plasticity in FID rats. Moreover, the current study suggests that interventions that enhance BDNF activity such as exercise or selective serotonin reuptake inhibitors (48, 49) may be useful as therapeutic approaches to treat long-term effects of fetal-neonatal iron deficiency.
We thank Dr. Raghavendra Rao for reviewing this manuscript and Heather McLaughlin for providing editorial assistance.
Financial Support: NICHD RO1 HD29421 to M.K.G, NIMH training grant T32MH073129 to P.V.T, and NINDS NRSA F31NS04876 to E.S.C.
Suggested Reviewers: Z. Leah Harris M.D. Associate Professor, International Health, John Hopkins University zharris1/at/jhmi.edu Related research interests
John D Barks M.D. Associate Professor, Department of Pediatrics and Communicable Diseases , University of Michigan jbarks/at/umich.edu Expertise in brain health and injury.
Cathy W Levenson Ph.D. Associate Professor, Program in Neuroscience, Florida State University levenson/at/neuro.fsu.edu Research related interests
Opposed Reviewers: John L Beard Ph.D. Professor, Graduate Program in Neuroscience, Penn State jbeard/at/psu.edu collaborator; conflict of interest
The authors have no financial interest or potential conflict of interest to disclose.