Prion Protein (PrP) Knock-Out Mice Show Altered Iron Metabolism: A Functional Role for PrP in Iron Uptake and Transport

Despite overwhelming evidence implicating the prion protein (PrP) in prion disease pathogenesis, the normal function of this cell surface glycoprotein remains unclear. In previous reports we demonstrated that PrP mediates cellular iron uptake and transport, and aggregation of PrP to the disease causing PrP-scrapie (PrPSc) form results in imbalance of iron homeostasis in prion disease affected human and animal brains. Here, we show that selective deletion of PrP in transgenic mice (PrPKO) alters systemic iron homeostasis as reflected in hematological parameters and levels of total iron and iron regulatory proteins in the plasma, liver, spleen, and brain of PrPKO mice relative to matched wild type controls. Introduction of radiolabeled iron (59FeCl3) to Wt and PrPKO mice by gastric gavage reveals inefficient transport of 59Fe from the duodenum to the blood stream, an early abortive spike of erythropoiesis in the long bones and spleen, and eventual decreased 59Fe content in red blood cells and all major organs of PrPKO mice relative to Wt controls. The iron deficient phenotype of PrPKO mice is reversed by expressing Wt PrP in the PrPKO background, demonstrating a functional role for PrP in iron uptake and transport. Since iron is required for essential metabolic processes and is also potentially toxic if mismanaged, these results suggest that loss of normal function of PrP due to aggregation to the PrPSc form induces imbalance of brain iron homeostasis, resulting in disease associated neurotoxicity.


Introduction
Prion protein (PrP) is a ubiquitously expressed cell surface glycoprotein linked to the plasma membrane by a glycosylphosphatidylinositol (GPI) anchor. Although several lines of evidence support the obligate role of PrP in animal and human prion disorders, relatively less is known about the normal function of this protein [1,2]. Attempts to understand the physiological function of PrP by generating PrP knock-out (PrP KO ) transgenic mouse lines have been futile since the mice do not develop an overt phenotype other than resistance to prion disease [3]. The limited information on possible physiological function(s) of PrP has been obtained from the Zurich 1 and Edinburgh PrP KO mice since these do not upregulate Doppel, a PrP homologue that induces cerebellar degeneration [4,5]. Most of the investigations on these and other mouse models have focused on brain function since PrP is most abundant on neuronal cells and is therefore likely to alter brain function by its absence. A similar loss of function due to aggregation of PrP to the disease causing PrP-scrapie (PrP Sc ) form is also likely to alter neuronal function, partially explaining the pathogenesis of prion disorders and justifying the focus [2]. Several important facts have emerged from these studies; PrP KO mice show altered circadian rhythm and sleep pattern, increased susceptibility to neuronal damage by oxidative stress and cerebral ischemia, neurotoxicity by expression of Doppel and N-terminally truncated PrP, increased predilection to seizures, motor and cognitive abnormalities, reduced synaptic inhibition and long term potentiation in the hippocampus, altered development of the granule cell layer, mis-regulation of the cerebellar network, and age-dependent spongiform change with reactive astrogliosis [5][6][7][8][9][10]. Examination of peripheral organ systems reveals impaired ability of hematopoietic progenitor cells of PrP KO mice to colonize when transplanted to irradiated recipient mice [11], and poor recovery of PrP lacking animals from experimentally induced anemia [12]. The multiplicity of observations attributed to the absence of PrP in the same transgenic mouse line suggests its involvement in an essential function with broad implications. Though attractive, this hypothesis has remained untested.
Recently, we demonstrated that PrP mediates iron uptake and transport in human neuroblastoma cells [13], and aggregation of PrP to the disease causing PrP-scrapie (PrP Sc ) form induces an imbalance of iron homeostasis in prion disease affected human, hamster and mouse brains [14]. Here, we extend the cell based studies to mouse models to understand the role of PrP in iron metabolism in vivo. Using the Zurich 1 PrP KO and matched wild type (Wt) mice expressing normal levels of PrP as experimental models, we demonstrate that PrP KO mice show a phenotype of systemic iron deficiency and altered iron homeostasis compared to Wt controls. The underlying cause of this abnormality is impaired transport of iron from the duodenal epithelium to the blood stream, and a similar defect in iron uptake by parenchymal cells of various organs and hematopoietic precursor cells. The iron deficient phenotype of PrP KO mice is rescued by re-introducing PrP on the PrP KO background, indicating a functional role for PrP in iron uptake and transport. Since iron is an essential element that is necessary for survival but can be extremely toxic if mis-managed [15], these results have significant implications for understanding the physiological function of PrP in cellular iron metabolism, and the pathological implications thereof due to its aggregation to the PrP Sc form, the principal agent responsible for prion disease associated neuronal death.

Results
Major organs of PrP KO mice show a phenotype of relative iron deficiency Since PrP is expressed most abundantly on neurons, the effect of PrP deletion on brain iron homeostasis was assessed in PrP KO and matched Wt controls. In iron deficiency, we expect low levels of total iron, decreased levels of iron storage protein ferritin, and increased levels of iron uptake proteins Tf and TfR in the affected tissue. To evaluate these parameters, equal quantity of protein from brain homogenates of PrP KO and Wt mice was fractionated by SDS-PAGE and immunoblotted ( Figure 1A). Probing for PrP reveals the expected glycoforms migrating between 27-37 kDa in Wt, and complete absence of PrP in PrP KO samples as expected.
Probing for ferritin shows a decrease in H-and L-chain isoforms in PrP KO samples relative to Wt controls. Tf and TfR, on the other hand, show a relative increase in PrP KO samples. Probing for bactin confirms that the observed differences are not an artifact of protein loading ( Figure 1A, lanes 1-6). Quantification by densitometry shows a decrease in ferritin levels by 59% and an increase in Tf and TfR levels by 58% and 245% respectively in PrP KO samples relative to Wt controls ( Figure 1B).
Brain homogenate represents a mixture of different cell types and the above results do not necessarily reflect the iron status of neurons, the cell population most vulnerable to alterations in iron homeostasis. To evaluate the iron status of neurons, unfixed brain sections from Wt and PrP KO mice were incubated with FITCtagged Tf for 15 minutes at 37uC [16], washed gently with cold PBS to remove unbound Tf, and fixed with paraformaldehyde. A set of control samples were pre-incubated with unlabeled apo-Tf for 15 minutes before exposure to FITC-Tf to compete for binding sites. Examination by fluorescence microscopy shows significantly more binding of FITC-Tf to the cerebellar Purkinje cell layer of PrP KO brain sections compared to Wt controls, suggesting higher expression of TfR in PrP KO neurons (Figure 2A, panels 1 and 2, white arrow). The binding of FITC-Tf is inhibited competitively by pre-incubation of brain sections with unlabeled apo-Tf, indicating the specificity of the reaction (Figure 2A, panels 3 and 4, white arrow-head). To further analyze the state of iron deficiency in PrP KO brains, equal counts of 59 FeCl 3 were injected in the tail vein of Wt and PrP KO mice, and after a chase of 3 hours, mice were euthanized and cold PBS was infused from the heart to drain capillary blood. Subsequently, brains were harvested, counted in a c-counter, and fixed overnight in formalin. Subsequently, 700 mM sections were cut and exposed to X-ray film. Brains of PrP KO mice take up 45% more 59 Fe relative to Wt controls as estimated by total counts and visualization on X-ray film exposed to dry sections ( Figure 2B, panels 1-4, black arrows). These results indicate a state of relative iron deficiency and consequent increased transport of 59 Fe to the brains of PrP KO mice.  Incubation of unfixed brain sections with FITC-Tf shows significantly more binding to the Purkinje cell neurons of PrP KO sample compared to Wt controls (panels 1 and 2) [16]. Pre-incubation of brain sections with unlabeled apo-Tf decreases the signal significantly in both samples (panels 3 and 4), demonstrating the specificity of binding. The assay was performed on three sections each from three different sets of mice. A representative section is shown. (B) Autoradiograph of brain sections prepared from Wt and PrP KO mice injected with 59 FeCl 3 intravenously shows significantly more 59 Fe in PrP KO sections compared to Wt controls (panels 1-4). A scan of brain sections exposed to the film in panels 2 and 4 respectively is shown (panels 5 and 6). (Two consecutive 700 mM sections from each brain sample are shown. 59 Fe is visible as radio-opaque black dots. doi:10.1371/journal.pone.0006115.g002 To determine whether a similar phenotype of iron deficiency is observed in other major organs of PrP KO mice, the iron content and levels of the iron management proteins ferritin and Tf were evaluated in the liver of Wt and PrP KO mice. Frozen sections of Wt and PrP KO liver were stained for iron with Prussian blue and examined. PrP KO sections show significantly less iron in Kupffer cells and hepatocytes relative to Wt controls ( Figure 3A, panels 1 and 2). Fractionation of liver homogenates on a non-denaturing gel followed by in-gel reaction with Ferene-S shows lower iron content in PrP KO liver ferritin compared to Wt controls ( Figure 3B, lanes 1-4) [17]. (The identity of the blue band as ferritin was established by immunoblotting as described previously [13]). Evaluation of ferritin and Tf expression in liver homogenates shows significantly lower levels of ferritin and higher levels of Tf in PrP KO samples compared to Wt controls ( Figure 3C, lanes 1-6). Quantification by densitometry shows a decrease in the iron content and expression of ferritin by 48% and 66% respectively, and an increase in the expression level of Tf by 44% in PrP KO samples relative to Wt controls ( Figure 3D).
A similar analysis of spleen sections shows intense reaction for iron with the Perl's stain in both Wt and PrP KO samples ( Figure 4A, panels 1 and 2). In-gel reaction of spleen ferritin with Ferene-S shows a decrease in the iron content and expression of ferritin in PrP KO samples relative to Wt controls (Figures 4B and C, lanes 1-6). Quantification by densitometry shows a decrease in the iron content and expression of ferritin by 27%, and 64% in PrP KO samples compared to Wt controls ( Figure 4D). Re-probing of the membrane for Tf and TfR did not show a significant difference in the expression of these proteins (data not shown).
Together, the above results suggest a phenotype of relative iron deficiency in the brain, liver, and spleen of PrP KO mice compared to Wt controls. Subsequent studies were directed at characterizing the iron deficient phenotype of PrP KO mice and the mechanistic basis of this finding.

The iron deficiency of PrP KO mice is largely compensated
To evaluate if the phenotype of iron deficiency in PrP KO mice is due to low circulating iron, hematological parameters were evaluated by an automated blood chemistry analyzer optimized for mouse samples. Hemoglobin, serum iron, Tf saturation, and serum ferritin are decreased in PrP KO samples by 3.5, 21.6, 37.6 and 50.1% respectively, while the reticulocyte count and total iron binding capacity (TIBC) are increased by 42.2 and 24.0% respectively in PrP KO samples relative to matched Wt controls ( Table 1). The total red cell count and hematocrit show minimal differences between the two groups. Bone marrow smears revealed an increase in red cell precursors in PrP KO samples, indicating an attempt by the iron homeostatic machinery to compensate for the iron deficiency in PrP KO animals (data not shown).
Subsequent studies were directed at determining whether the iron deficiency in PrP KO mice arises from inefficient uptake and/ or transport of iron from ingested food, defective uptake by target PrP mediates iron uptake and/or transport from the duodenum to the blood stream The uptake and transport of ingested iron from the intestinal lumen to the blood stream and target organs of Wt and PrP KO mice was evaluated by introducing radioactive iron into the stomach, and quantifying 59 Fe uptake by various organs after defined time points of chase. The following organs were evaluated: 1) stomach with attached duodenum, jejunum, and a small piece of ileum, 2) liver, 3) spleen, 4) brain, 5) femurs, and 6) tibial bones. Washed upper gastrointestinal tract was vacuum-dried and exposed to X-ray film ( Figure 5A), and after obtaining adequate film exposures, 59 Fe incorporated in the upper 10 cm of the duodenum was quantified in a c-counter. Whole blood, washed red blood cells (RBCs), plasma, organs, and bones were counted in a c-counter to quantify 59 Fe incorporation.
Autoradiographs of duodenum samples demonstrate significantly more 59 Fe in PrP KO samples at each time point compared to Wt  controls ( Figure 5A, lanes [1][2][3][4][5][6][7][8][9][10][11][12]. Surprisingly, the amount of 59 Fe in the duodenum of PrP KO mice is significantly higher than Wt samples even after 11 days of chase ( Figure 5A, lanes 11 and 12). Samples in lanes 1-8 were exposed to X-ray film for 2 hours, lanes 9 and 10 for 24 hours, and lanes 11 and 12 for 6 days to highlight the difference in 59 Fe content between Wt and PrP KO samples ( Figure 5A, lanes 9-12). Quantitative analysis of these results was performed by counting the first 10 cm of the duodenum from each sample in a c-counter. As expected, both samples show a gradual decline in 59 Fe counts with increasing chase time, falling to 0.1% of the initial value after 11 days of chase. However, PrP KO samples show higher retention of 59 Fe by 21, 60, 87, 32, and 57% relative to matched Wt controls after a chase of 1, 4, 24, and 48 hours and 11 days respectively ( Figure 5B). Mice ranging in age from 3-6 months and blinded to the person performing the experiment yielded similar results. The 1 and 4 hour chase time points were repeated more than 6 times. To evaluate whether increased retention of 59 Fe in the duodenum of PrP KO mice is due to sequestration in ferritin within enterocytes, 59 FeCl 3 fed Wt and PrP KO mice were chased for 4 hours, and the first 5 cm of the duodenum was homogenized and fractionated on a non-denaturing gel to identify 59 Fe-labeled proteins as described previously [13]. A single iron labeled band that immunoreacts for ferritin is detected in Wt and PrP KO samples ( Figure 6A, lanes 1-4). The iron content and expression of ferritin are higher in the PrP KO sample compared to Wt control, explaining the significantly higher 59 Fe content in the duodenum of PrP KO mice ( Figure 6A, lanes 1-4). Fractionation of the same samples by SDS-PAGE followed by immunoblotting confirms that ferritin levels are significantly higher in the PrP KO sample ( Figure 6A, lanes 5 and 6). (The identity of iron labeled bands in lanes 1 and 2 was confirmed by eluting labeled proteins from these bands and re-fractionating on SDS-PAGE followed by immunoblotting as described previously [13]). Quantification by ccounting and densitometry shows an increase in the 59 Fe content of ferritin by 42% and expression of ferritin by 119% in PrP KO samples relative to matched Wt controls ( Figure 6B).
These results indicate that PrP KO mice take up more 59 Fe from the intestinal lumen and/or release less into the blood stream compared to Wt controls, resulting in the accumulation of 59 Fe in the duodenal epithelium.

PrP facilitates iron uptake by parenchymal cells and hematopoietic precursor cells
Transport of iron from the duodenal epithelium to the blood stream and subsequent uptake by various organs was evaluated by quantifying 59 Fe in the plasma, washed RBCs, and various organs in a c-counter, and fractionating the plasma using a nondenaturing gel to evaluate the 59 Fe content of Tf. The 59 Fe counts in the plasma of PrP KO mice are decreased by 12% after 1 hour, and increased by 47, 108, 51, and 40% after 4, 24, and 48 hours and 11 days of chase respectively relative to Wt controls (Figure 7, plasma). Red blood cells show no incorporation of 59 Fe after 1 hour as expected, a sudden increase in PrP KO samples by 537% after 4 hours, and a decrease by 60, 48, and 63% after 24 and 48 hours and 11 days respectively relative to Wt controls ( Figure 7, red cells). The spleen of PrP KO mice shows 62% less 59 Fe after 1 hour, an increase by 831% after 4 hours, followed by a precipitous fall by 64% of Wt values after 24 hours that is maintained throughout 11 days of chase (Figure 7, spleen). Likewise, the femurs of PrP KO mice show 25% less 59 Fe after 1 hour, an increase by 261 and 17% after 4 and 24 hours, and a decline by 39% after 11 days respectively relative to Wt controls. Femurs of Wt mice also show a spike in 59 Fe after 24 hours of chase, but the increase is significantly less than PrP KO samples (Figure 7, Femur). The liver of PrP KO mice shows an increase in 59 Fe by 47 and 82% after 1 and 4 hours, an increase by 339% after 24 hours, and a precipitous fall by 69% after 11 days of chase relative to Wt controls (Figure 7, liver). Brain samples show variability during the first 48 hours of chase in both PrP KO and Wt mice, but after 11 days there is a significant decrease in PrP KO samples by 69% of Wt values (Figure 7, brain).
Fractionation of plasma by non-denaturing gel electrophoresis followed by autoradiography reveals the presence of 59 Fe-Tf in Wt and PrP KO samples as expected ( Figure 8A) (The identity of 59 Fe labeled band as Tf was established by immunoblotting as reported earlier [13]). The 59 Fe content of Tf in the PrP KO sample is lower after 1 hour and significantly higher after 4 and 24 hours of chase relative to Wt controls ( Figure 8A, lanes 1-6). Minimal signal is detected after 48 hours of chase in either of the samples ( Figure 8A, lanes 7 and 8). Evaluation of the 59 Fe content of RBCs spotted on a PVDF membrane followed by autoradiography shows increased incorporation in PrP KO samples after 4 hours and minimal increase thereafter until 48 hours of chase. Wt samples, on the other hand, show significant incorporation after 24 hours and almost doubling of the counts by 48 hours (Figure 8B), reflecting the values in Figure 7 above. These results indicate inefficient transport of 59 Fe from the duodenum of PrP KO mice to the blood stream, but much faster kinetics of incorporation into RBC precursors by 4 hours of chase and minimal increase thereafter. In contrast, Wt mice show significantly higher transport of 59 Fe to the blood stream, incorporation into RBCs at the expected chase time of 24 hours, and a linear increase in the 59 Fe content of RBCs thereafter. Since the majority of Tf-bound 59 Fe is taken up by newly synthesized RBCs, the increase in plasma 59 Fe-Tf in PrP KO mice after 4 hours of chase relative to Wt controls indicates decreased uptake as evidenced by the lower 59 Fe content of PrP KO RBCs ( Figure 8A, lanes 3-6).
Together, the above results indicate that the transport of iron from the duodenal enterocytes to the blood stream and uptake by  Figure 5 above were homogenized and separated on a non-denaturing gel followed by autoradiography (lanes 1 and 2). The level of 59 Fe labeled ferritin is significantly higher in the PrP KO sample compared to matched Wt control (lanes 1 and 2). Transblotting under native conditions followed by immunoblotting for ferritin confirms the identity of 59 Fe labeled bands as ferritin (lanes 3 and 4). Fractionation of the same samples by SDS-PAGE followed by immunoblotting shows relatively more ferritin in the PrP KO sample compared to matched Wt control (lanes 5 and 6). the RBCs, liver, and brain is less efficient in PrP KO mice and follows different kinetics of utilization relative to Wt controls. It is likely that PrP KO mice make an early attempt at erythropoiesis because of their iron deficient state, but are unable to incorporate enough iron into RBCs due to defective uptake by hematopoietic progenitor cells. The lower 59 Fe content of PrP KO RBCs is not due to a shorter life-span of these cells since their 59 Fe counts remain constant throughout 11 days of chase (Figure 7), the normal halflife of murine RBCs. No signs of hemolysis were detected at any time point of chase by urine-analysis (data not shown).
Iron deficient phenotype of PrP KO mice is not due to sequestration of iron in reticulo-endothelial cells To rule out low level of hemolysis combined with sequestration of 59 Fe in cells of the reticulo-endothelial system in PrP KO mice, spleens harvested after 1-48 hours of chase (as in Figure 7 above) were homogenized, and equal quantity of protein was fractionated by non-denaturing gel electrophoresis followed by autoradiography. Prominent bands of 59 Fe-ferritin and 59 Fe-Tf are detected in the PrP KO sample after 4 hours of chase revealing 59 Fe content several-fold higher than matched Wt controls (Figure 9, lanes 3 and 4). However, the signal is lost by 24 hours of chase, making it unlikely that reticulo-endothelial cells sequester the incorporated 59 Fe (Figure 9, lanes 5-8). At all other time points the Wt sample shows slightly higher 59 Fe-ferritin and 59 Fe-Tf compared to PrP KO samples ( Figure 9, lanes 1, 2, and 5-8). The identity of 59 Fe labeled bands as ferritin and Tf was established by immunoblotting under native conditions as described previously [13].
Further confirmation of the above results was obtained by culturing peritoneal macrophages from Wt and PrP KO mice overnight, loading them with 59 FeCl 3 -citrate complex after stimulation with LPS [18], and monitoring release of 59 Fe into the medium in a c-counter. Macrophages from PrP KO mice release 17% of intracellular iron into the medium compared to 13% from the Wt sample during a chase of 18 hours, making it unlikely that sequestration of 59 Fe in the reticulo-endothelial cells is responsible for the iron deficient phenotype of PrP KO mice ( Table 2).

Expression of wild type PrP rescues the iron deficient phenotype of PrP KO mice
Since the only difference between Wt and PrP KO mice is the absence of PrP expression in the latter, a single allele of Wt PrP was re-introduced on the PrP KO background to generate PrP + mice. To assess the expression levels of PrP in PrP + mice relative to Wt controls, an equal quantity of protein from brain homogenates  of Wt, PrP KO , and PrP + mice was fractionated by SDS-PAGE and immunoblotted. Probing for PrP shows the expected glycoforms of PrP migrating between 27-37 kDa in Wt and PrP + samples ( Figure 10A, lanes 1 and 3). Quantification by densitometry shows slightly higher PrP expression in PrP + mice, ,10% relative to Wt controls. PrP KO samples do not react for PrP as expected ( Figure 10A, lane 2). Thus, PrP + mice are a suitable model to test whether expression of PrP rescues the iron deficient phenotype of PrP KO mice.
To reduce variability, littermate PrP KO and PrP + mice were obtained by crossing PrP + and PrP KO breeding pairs. After confirming the genotype (data not shown), littermate PrP + and PrP KO , Wt, and PrP KO mice were evaluated for iron uptake and transport by introducing equal amounts of 59 FeCl 3 by gastric gavage as in Figures 5 and 7 above (Figure 10B and C). Only the 4 hour chase time point was assessed since the maximum differences between Wt and PrP KO mice are observed at this time point ( Figure 5 and 7 above). Autoradiography of duodenum samples shows significantly more 59 Fe in the PrP KO sample compared to the Wt control as noted in Figure 5 above ( Figure 10B, lanes 1 and 2). Notably, a similar difference is seen between PrP KO and PrP + littermates ( Figure 10B, lanes 3 and 4),   2 and 4). Notably, the 59 Fe content of PrP (+) duodenum is similar to Wt, and significantly less than the littermate PrP KO sample (lanes 1, 3 and 4) indicating improved transport of 59 Fe from the duodenum of mice expressing PrP. Wt and PrP + samples show similar levels of iron, confirming the above results ( Figure 10A, lanes 1 and 3). Quantification of 59 Fe in the first 10 cm of duodenum samples shows an increase in PrP KO samples by 132% relative to Wt controls, and an increase in littermate PrP KO by 238% relative to littermate PrP + controls ( Figure 10C). As expected, Wt and PrP + samples show minimal difference in their 59 Fe content ( Figure 10C, lanes 1 and 3). Quantification of 59 Fe in the blood, liver, spleen and femur shows similar incorporation in Wt and PrP + controls, and higher levels in PrP KO mice as observed in Figure 7 above (Figure 11). These observations demonstrate that the deficiency of iron uptake in PrP KO mice is reversed by the expression of PrP in the PrP + mice.

Discussion
In this report, we demonstrate that the absence of PrP induces systemic iron deficiency in PrP KO mice, a phenotype that is rescued by re-introducing PrP on the PrP KO background. Relative to normal Wt mice of the same genetic background, PrP KO mice exhibit lower levels of iron in the plasma, brain, liver, and spleen, and the major iron metabolism proteins show a compensatory response by increasing the levels of iron uptake proteins Tf and TfR, and decreasing the levels of iron storage protein ferritin. The underlying cause of iron deficiency in PrP KO mice is likely to be two-fold; 1) inefficient transport from duodenal enterocytes to the blood stream, and 2) impaired uptake by target cells of various organs, including the hematopoietic precursor cells. We previously reported that PrP functions as an iron uptake and transport protein in human neuroblastoma cells in vitro [13]. This report supports and extends those findings by demonstrating a similar function of PrP in mouse models, a novel function for a protein known mostly for its role in the pathogenesis of prion disorders.
It is surprising that the mere absence of PrP alters iron metabolism in both enterocytes and hematopoietic cells since distinct processes regulate iron uptake and transport at these sites. In the intestine, absorption of non-heme iron, the form used in this study, is mediated by the divalent metal transporter 1 (DMT1) at the apical (AP) plasma membrane of duodenal epithelial cells. Here, duodenal cytochrome b (DcytB) functions as a ferric reductase and reduces Fe(III) iron to the Fe(II) form for transport to the cytosol by DMT1. Absorbed iron is transported through the cytosol by poorly characterized mechanisms and is exported from the basolateral (BL) membrane by the concerted effort of ferroportin (FPT) and hephaestin, a multicopper oxidase that oxidizes Fe(II) iron to the Fe(III) form for uptake by Tf in the systemic circulation [19].
Based on our observations, it appears that PrP mediates iron export from the BL membrane of enterocytes to the blood stream rather than uptake from the intestinal lumen. This conclusion is supported by our observations demonstrating accumulation of orally administered radioactive iron ( 59 FeCl 3 ) in the duodenal enterocytes of PrP KO mice that persists until eleven days of chase when almost all 59 Fe from Wt controls has been transported out. Most of the 59 Fe accumulates in ferritin within these cells, a surprising observation given the state of iron deficiency in these mice. Ferritin protein expression is also up-regulated in PrP KO enterocytes, probably reflecting a response to increased iron influx from the AP plasma membrane, decreased transport from the BL membrane, or a combination of the two processes. Although limited amounts of 59 Fe are released into the blood stream of PrP KO mice during the first hour of chase, the amount is significantly lower compared to Wt controls. Furthermore, PrP is expressed on the BL surface of MDCK cells, a polarized epithelial cell line, and inter-cellular junctions of mouse enterocytes [20,21], suggesting a functional role at the BL domain rather than the AP plasma membrane. It is surprising that PrP KO mice excrete relatively less 59 Fe in their feces despite the iron loaded state of their enterocytes (unpublished observations). It is likely that this response reflects an attempt to conserve and utilize the absorbed iron by PrP KO mice due to their chronic state of iron deficiency.
A similar phenotype of systemic iron deficiency resulting from accumulation of absorbed iron in the duodenal epithelium has been reported in sex-linked anemia (Sla) mice due to a mutation in the intestinal ferroxidase Hephaestin [22]. Based on our data, it is likely that PrP functions upstream from hephaestin, perhaps as a ferric-reductase, to reduce Fe(III) iron stored in ferritin to the Fe(II) form for transport across the BL membrane and subsequent oxidation by hephaestin for delivery to circulating Tf. It is interesting to note that PrP has been reported to perform a similar function in copper transport where it reduces copper (II) prior to its delivery to cytosolic copper (I) carrying proteins [23]. It seems unlikely that PrP is an active ferroxidase based on a previous report [13] and our current observations on the rate of iron efflux from peritoneal macrophages of PrP KO mice. However, differential activity of PrP in different cell lines cannot be ruled out, leaving it unclear whether PrP indeed functions as a ferroxidase like hephaestin. Since the iron deficiency in PrP KO mice is largely compensated and the animals live normally except for delayed erythropoiesis after induced hemolysis and specific deficiencies restricted mainly to the central nervous system [5,12], it is likely that PrP modulates the function of other iron uptake proteins or is involved in a parallel pathway that compensates for its absence. Identification of such proteins and pathways would be a significant step forward in understanding the role of PrP in iron export from the duodenum.
Absence of PrP also compromises the ability of hematopoietic precursor cells to take up iron from plasma Tf, an interesting observation since these cells acquire most of their iron by the Tf/ TfR pathway. A similar phenotype of iron deficiency has been reported in neuroblastoma cells expressing lower levels or mutant forms of PrP, another cell line that utilizes the Tf/TfR pathway for acquiring iron [13]. It is therefore likely that PrP functions at a point where the enterocyte and the Tf/TfR pathway of iron uptake and transport intersect. Normally, plasma Tf provides iron to most cells of the body, and itself receives iron from three main sources; 1) food via the duodenum and proximal jejunum, 2) recycled senescent RBCs, and 3) iron stores within macrophages and hepatocytes. Most of the iron taken up from food is utilized for erythropoiesis, and RBCs contain 80% of the total body iron. Iron rich Tf binds to the TfR on hematopoietic precursor cells, and the Tf/TfR complex is endocytosed through clathrin mediated endocytosis. Tf bound iron is released in the acidic environment of the endosomes, reduced by the ferric reductase Steap3, and transported across the membrane by DMT1 to cytosolic ferritin for storage. Ferritin maintains the cellular labile iron pool to provide for the metabolic needs of cells, and is itself regulated by the iron content of cells through iron management proteins [22][23][24][25][26]. It is surprising that the mere absence of PrP compromises the ability of parenchymal cells of various organs and hematopoietic precursor cells to take up iron.
PrP KO mice show an earlier and a significantly higher spike in 59 Fe uptake by the spleen and long bones relative to Wt controls. Since RBCs consume the majority of plasma iron and the spleen and long bones are the principal sites of erythropoiesis, these findings reflect an abnormally high rate of erythropoiesis in PrP KO mice. The presence of 59 Fe labeled cells in the peripheral blood of PrP KO mice after four hours of chase is surprising given the time it takes for RBCs to mature, and may reflect the binding of 59 Fe-Tf to TfR positive immature erythrocytes. However, while Wt RBCs continue to incorporate 59 Fe, PrP KO RBCs show only a modest increase even after eleven days of chase. These results suggest that the erythropoiesis in PrP KO mice is ineffective despite adequate supplies of iron in the spleen and long bones, probably due to inefficient uptake by the hematopoietic precursor cells. The alternate possibility, i.e., increased turnover of RBCs in the spleen or intravascular hemolysis is unlikely based on the following observations: 1) 59 Fe counts did not increase in the spleen of PrP KO mice at any time point after four hours of chase, the main organ where RBCs are turned over and the released iron recycled, 2) PrP KO RBCs did not show a decline in 59 Fe counts during eleven days of chase given that the normal half-life of mouse RBCs is ,15 days, 3) 59 Fe from hemolyzed RBCs was not detected in the urine, and 4) markers of hemolysis such as hemoglobinuria were not detected in PrP KO mice (unpublished observations). Furthermore, the kinetics of 59 Fe uptake from the plasma of Wt and PrP KO mice suggests decreased uptake by target organs rather than increased hemolysis in the latter. The precise biochemical pathway by which PrP facilitates iron uptake by the parenchymal and hematopoietic precursor cells is difficult to define from our data. However, based on our observations from neuroblastoma cells [13], it is likely that PrP facilitates transport across the endosomal membrane to the cytosol by functioning as a ferric reductase. Further studies are required to resolve this question.
The iron deficiency in PrP KO mice is mild since overt signs of anemia were not detected except for a mild reduction in hemoglobin levels, a significant reduction in serum iron, serum ferritin, and transferrin saturation, and an increase in the total iron binding capacity and the number of circulating reticulocytes. This observation is surprising given the obvious decrease in brain iron content, a privileged organ that is affected last by iron deficiency [27]. It is likely that the response of iron management proteins compensates for the iron deficiency in PrP KO mice in all major organs, including the hematopoietic system. Introduction of PrP on the PrP KO background reverses the iron deficient phenotype completely, indicating that PrP is an integral component of the iron homeostatic machinery that feeds into the major pathways of iron uptake and transport.
In conclusion, this report demonstrates a significant role of PrP in maintaining systemic iron homeostasis. Specifically, PrP modulates iron transport from the duodenum to the blood stream and uptake by cells of the hematopoietic system, brain, liver, and spleen. Since major proteins and pathways of iron transport and uptake utilized by enterocytes and hematopoietic cells differ significantly [26], these observations suggest a functional role for PrP at a point downstream from these pathways. Although our data fall short of identifying the specific role of PrP in iron modulation, this report establishes the functional role of PrP in iron metabolism, and provides essential information on the possible sites where PrP influences iron metabolism independently, or by intersecting with known pathways of iron uptake and transport. Since imbalance of iron homeostasis is a common feature of prion disease affected human, hamster, and mouse brains [14], these results suggest a significant contribution of loss of PrP function to prion disease pathogenesis.

Materials and antibodies
PrP-specific monoclonal antibody 8H4 (against residues 145 to 180) was obtained from Abcam (Cambridge, MA, USA) and from Drs. Man-Sun Sy and Pierluigi Gambetti (Case Western Reserve University). Rabbit anti-human ferritin antibody that cross-reacts with H and L-chains of mouse ferritin was obtained from Sigma (catalog # F5012, lot number 117K4880), ferritin H-chain specific antibody was from Santa Cruz (Catalog # sc-25617, lot # L0104), ferritin L-chain specific antibody was also from Santa Cruz (Catalog # sc-25616, lot # E0905), anti-Tf was from GeneTex (San Antonio, TX), anti-TfR from Zymed Laboratories Inc (Carlsbad, CA), and horseradish peroxidase (HRP)-conjugated secondary antibodies were from GE Healthcare (Little Chalfont, Buckinghamshire, United Kingdom). All other chemicals were purchased from Sigma.

Transgenic mice
The PrP KO mice in the FVB background (FVB/Prnp 0/0 ) were originally obtained from George Carlson, (McLaughlin Research Institute). The PrP + transgenic mice expressing wild type murine PRNP were created by microinjection of the half-genomic PrP clone [28] into fertilized FVB/Prnp 0/0 eggs. The transgenic mice were screened by PCR and maintained via breeding with FVB/ Prnp 0/0 mice. The genotype of littermate PrP KO and PrP + mice were determined by PCR of DNA from ear punches.

Intestinal iron uptake and transport
All procedures with mice were performed according to the guidelines established by the Animal Resource Center of Case Western Reserve University, and were based on protocols approved by the IACUC committee. Three to six month old age-and sex-matched Wt and PrP KO mice maintained under similar conditions were fasted overnight with water ad libitum, and 20 mCi of 59 FeCl 3 diluted in 0.2 ml of PBS was administered orally with an olive-tipped gavage needle. The mice were chased on normal chow for 1 h, 4 h, 24 h, 48 h and 11 days. At each chase time point, a set of Wt and PrP KO mice were euthanized, and blood was collected by cardiac puncture in heparinized vials. Plasma and red cells were separated by centrifugation at 5,000 rpm for 5 min. Stomach and upper gastro-intestinal tract was separated and rinsed with cold PBS until clean. The samples were placed on a filter paper, vacuum dried, and subjected to autoradiography. The brain, liver, spleen, and femur were collected, washed with PBS, and snap frozen on dry ice. The organs were weighed, and radioactivity was counted in a ccounter. Selected samples of brain were fixed in 5% phosphate buffered formalin for 24 h, and 700 mM thick sections were cut using a Leica vibrotome. Cut sections were briefly air-dried and exposed to X-ray film.
Native gradient gel electrophoresis, autoradiography and immunoblotting Native gradient gel electrophoresis, autoradiography, and immunoblotting were performed essentially as described by Vyoral et al. [29] and in a previous report [13].

SDS-PAGE and Western blotting
Brain, liver, and spleen homogenates (10%) prepared in lysis buffer were resolved by SDS-PGE and subjected to western blotting using specific antibodies as described in previous reports [30]. Immunoreactive bands were visualized by ECL detection system (Amersham Biosciences Inc.).

Prussian blue staining
Snap frozen liver and spleen sections (7 mm) were immersed in acidified potassium ferrocyanide solution (4%) for 20 min followed by washing with distilled water. Sections were then counterstained with 1% Neutral Red for 2 min and mounted.

Determination of plasma and tissue iron
In-gel iron estimation of protein bands and the iron content of liver and spleen homogenates were performed by Ferene-S staining essentially as described [14]. Serum iron, total iron binding capacity and transferrin saturation were determined by using an automated Ferene based detection method on a Dimension RXL chemistry analyzer (Dade Behring, Deerfield, Ill). The CBC and reticulocyte data was determined on a Sysmex XE-2100 hematology analyzer (Kobe, Japan). To detect hemolysis, urine dip-stick was performed on an Urisys 2400 urine analyzer (Roche Diagnostics, Indianapolis IN).

Transferrin binding assay
Tf binding sites in the brains of Wt and PrP KO mice were assessed by incubating 10 mM thick brain sections prepared from frozen samples essentially as described by Moos et al. [17]. In short, brain sections were pre-incubated with 0.2% bovine serum albumin in PBS for 30 min at 37uC in a humid atmosphere to wash off endogenous Tf, and incubated with FITC-Tf (5 mg/ml) for 30 min at 37uC in a humid chamber. Another set of samples was pre-incubated with apo-transferrin (75 ng) for 10 min at room temperature followed by incubation with FITC-Tf as above. The sections were rinsed with PBS, mounted, and observed using a laser scanning confocal microscope (Bio-Rad MRC 600).

Estimation of iron export from peritoneal macrophages
Peritoneal macrophages were isolated from Wt and PrP KO mice as described by Hanazawa et al. [18]. Following an overnight culture, adhered macrophages were stimulated with LPS (100 ng/ ml) for 24 h, serum starved for 1 h, and labeled with 59 FeCl 3citrate complex (1 mM sodium citrate and 2 mCi of 59 FeCl 3 in serum free DMEM for 4 h at 37uC. The molar ratio of citrate to iron was maintained at 100:1. Cell surface bound iron was washed and the cells were chased in complete medium for different time periods. A 100 ml aliquot of the medium was retrieved at each time point and counted in a c-counter. After 18 h the cells were lysed and cell associated 59 Fe was measured in a c-counter.

Statistical analysis
A minimum of 3 mice in each group were analyzed for all experiments, and the experiments were repeated at least 3 times. The results are expressed as mean6standard error of mean (SEM). Statistical analysis was done by unpaired Student's t test when comparing Wt with PrP KO group. For comparing Wt, PrP KO and littermates groups, one way ANOVA followed by Bonferroni multiple comparison post hoc test was done using GraphPad Prism software (Version 5.02, GraphPad Inc., San Diego, CA, USA). Differences were considered significant at p,0.05.

Author Contributions
Conceived and designed the experiments: NS. Performed the experiments: AS XL NS. Analyzed the data: AS HM NS. Contributed reagents/ materials/analysis tools: QK RP NS. Wrote the paper: NS. Prepared figures, wrote part of the paper: AS.