Time course of ⁵⁹Fe distribution in the blood of Wt and PrP^KO mice.

<p>(A): Equal volume Wt and PrP^KO plasma collected at different time points of chase were separated on a non-denaturing gel followed by autoradiography. After 1 hour of chase, ⁵⁹Fe-Tf in the PrP^KO sample is lower than matched Wt controls (lanes 1 and 2). However, the ratio reverses thereafter, and the level of ⁵⁹Fe-Tf in the PrP^KO sample is higher than Wt controls after 4, 24, and 48 hours of chase (lanes 3–8). (B) RBCs of Wt mice show minimal levels of ⁵⁹Fe after 1 and 4 hours, and significant increase after 24 and 48 hours of chase. PrP^KO mice, on the other hand, show iron counts at 4 hours, and minimal increase thereafter.</p

Iron is retained within ferritin in the duodenal epithelium of PrP^KO mice.

<p>(A) The 4 hour duodenum samples in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006115#pone-0006115-g005" target="_blank">Figure 5</a> above were homogenized and separated on a non-denaturing gel followed by autoradiography (lanes 1 and 2). The level of ⁵⁹Fe 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 ⁵⁹Fe 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). (B) Quantification by densitometry shows significantly more ⁵⁹Fe-ferritin and ferritin protein levels in the duodenum of PrP^KO mice relative to matched Wt controls. Values are mean±SEM, n = 3. *<i>p</i><0.001 relative to Wt.</p

Distribution of ⁵⁹Fe in PrP⁽⁺⁾ and PrP^KO litter-mates.

<p>Distribution of ⁵⁹Fe in the blood, liver, spleen, and femur after 4 hours of chase shows similar incorporation in Wt and PrP⁽⁺⁾ mice in the organs tested, and significantly more ⁵⁹Fe in the spleen and femur of PrP^KO mice as noted above. One way ANOVA shows a significant difference among Wt, PrP^KO, and PrP⁽⁺⁾ samples (p<0.0001). Post hoc Bonferroni tests show significant difference in ⁵⁹Fe in littermates PrP⁽⁺⁾ and PrP^KO organs relative to Wt controls (*p<0.01; **p<0.05). Littermate PrP⁽⁺⁾ shows a significant difference compared to PrP^KO counterpart (#p<0.01). Values are mean±SEM of three independent experiments.</p

Brain homogenates of PrP^KO mice show evidence of iron deficiency.

<p>(A) Immunoblot analysis of Wt and PrP^KO brain homogenates with PrP specific antibody 8H4 shows the expected glycoforms of PrP in Wt samples migrating between 27–37 kDa, and no reaction with PrP^KO samples as expected (lanes 1–6). Immunoreaction for ferritin, Tf, and TfR shows significantly less ferritin, and more Tf and TfR levels in PrP^KO samples compared to Wt controls (lanes 1–6). Reaction for β-actin confirms similar loading of protein. (B) Quantitative estimation of proteins in (A) after normalization with β-actin shows a decrease in ferritin, and an increase in Tf and TfR levels in PrP^KO samples compared to Wt controls. Values are mean±SEM. *<i>p</i><0.001, **<i>p</i><0.01 relative to Wt. n = 6.</p

Hematological parameters in Wt and PrP^KO mice.

<p>Values are mean±SEM. ^*<i>p</i><0.001, ^**<i>p</i><0.01, ^***<i>p</i><0.05 as compared to Wt. NS: not significant; N: number of animals in each group; RBCs: red blood cells; TIBC: total iron binding capacity; Tf: transferrin; ↓: decrease; ↑: increase.</p

Iron deficiency in PrP^KO mice is reversed by expressing Wt PrP.

<p>(A) Immunoblot analysis of Wt and littermate PrP^KO and PrP⁽⁺⁾ brain homogenates for PrP shows the expected glycoforms of PrP in Wt and PrP⁽⁺⁾ samples (lanes 1 and 3), and no reaction in the PrP^KO sample as expected (lane 2). (B) Autoradiogram of the upper gastrointestinal tract of Wt and littermate PrP⁽⁺⁾ and PrP^KO mice fed ⁵⁹FeCl₃ by gastric gavage as above and chased for 4 hours shows significantly more ⁵⁹Fe in the PrP^KO sample relative to Wt control (lanes 2 and 4). Notably, the ⁵⁹Fe content of PrP⁽⁺⁾ duodenum is similar to Wt, and significantly less than the littermate PrP^KO sample (lanes 1, 3 and 4). (C) Quantification of ⁵⁹Fe in the first 10 cm of the duodenum shows significantly more ⁵⁹Fe in the PrP^KO sample compared to matched Wt control. Similarly, more ⁵⁹Fe is detected in the PrP^KO sample compared littermate PrP⁽⁺⁾ control. One way ANOVA shows a significant difference in ⁵⁹Fe incorporation between Wt and PrP^KO, Wt and littermate PrP^KO, PrP⁽⁺⁾ and littermate PrP^KO, and PrP^KO and PrP⁽⁺⁾ (<i>p</i><0.0001). Post hoc Bonferroni tests show significant difference in ⁵⁹Fe in PrP^KO as well as littermate PrP⁽⁺⁾ and PrP^KO duodenums compared to Wt control (*<i>p</i><0.01; #<i>p</i><0.05). PrP⁽⁺⁾ exhibit significant difference compared to PrP^KO ($<i>p</i><0.01), and littermate PrP⁽⁺⁾ and PrP^KO also differ significantly (@p<0.01). Values are mean±SEM, n = 3.</p

Spleen of PrP^KO mice displays a phenotype of iron deficiency.

<p>(A) Prussian blue staining of frozen sections shows a prominent reaction in the reticulo-endothelial cells of PrP^KO and Wt samples. (B) In-gel reaction of homogenates with Ferene-S shows less iron content of PrP^KO ferritin compared to Wt controls (lanes 1–6). (C) Immunoreaction of homogenates with L-chain specific anti-ferritin antibody shows significantly less expression in PrP^KO samples compared to Wt controls (lanes 1–6). Re-probing for β-actin confirms that the difference is not an artifact of protein loading (lanes 1–6). (D) Quantification by densitometry after normalization with β-actin shows a significant reduction in the iron content and expression of ferritin in PrP^KO samples compared to matched Wt controls. Values are mean±SEM, n = 3. *<i>p</i><0.01 relative to Wt.</p

Distribution of ⁵⁹Fe in Wt and PrP^KO mice.

<p>Plasma: ⁵⁹Fe in PrP^KO samples is lower after 1 hour, increases by 48 hours, and falls to values below Wt after 11 days of chase. RBCs: Wt RBCs show ⁵⁹Fe incorporation after 24 hours, and the amount increases steadily till 11 days of chase. PrP^KO RBCs, on the other hand, incorporate ⁵⁹Fe relatively sooner after 4 hours, and show only a modest increase till 11 days of chase. Except for the 4 hour chase time, the amount of ⁵⁹Fe in PrP^KO RBCs is significantly lower than Wt controls, especially after 11 days of chase. Spleen: Wt samples show a slight increase in ⁵⁹Fe after 24 hours, and minimal change thereafter. PrP^KO spleens, on the other hand, show a significant increase after 4 hours, followed by a precipitous fall after 24 hours. Femur: Kinetics of ⁵⁹Fe uptake mirrors that of the spleen. PrP^KO samples show a spike at 4 hours that is maintained till 24 hours, followed by a fall. Wt samples show a delayed peak at 24 hours, and a fall to similar levels as the PrP^KO sample after 11 days. Liver: Wt samples show a steady increase in ⁵⁹Fe till 11 days of chase. PrP^KO samples, on the other hand, show a peak at 24 hours, and the counts fall thereafter to values significantly lower than Wt controls. Brain: Wt and PrP^KO samples do not show much difference at early time points of chase, but ⁵⁹Fe incorporation in the PrP^KO sample is significantly lower than Wt controls after 11 days of chase. Values are mean±SEM of three independent experiments. *<i>p</i><0.01, **<i>p</i><0.001 relative to Wt.</p

Brain sections of PrP^KO mice show up regulation of TfR and increased transport of iron from the blood stream.

<p>(A) 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) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0006115#pone.0006115-Moos2" target="_blank">[16]</a>. 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 ⁵⁹FeCl₃ intravenously shows significantly more ⁵⁹Fe 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 µM sections from each brain sample are shown. ⁵⁹Fe is visible as radio-opaque black dots.</p

Liver of PrP^KO mice displays a phenotype of iron deficiency.

<p>(A) Prussian blue staining of frozen sections shows minimal reaction for iron in the PrP^KO sample compared to Wt controls. (B) In-gel reaction of homogenates with Ferene-S shows significantly less iron content of PrP^KO ferritin compared to Wt controls (lanes 1–4). (C) Immunoreaction of homogenates with anti-ferritin antibody specific for H and L chains shows significantly less ferritin expression in PrP^KO samples compared to Wt controls (lanes 1–6). Re-probing for Tf shows an increase in Tf expression in PrP^KO samples relative to Wt controls (lanes 1–6). Levels of β-actin are similar in all samples, confirming that the observed differences are not an artifact of protein loading (lanes 1–6). (D) Quantification by densitometry shows a significant reduction in the iron content and expression of ferritin, and an increase in Tf expression in PrP^KO samples compared to matched Wt controls. Values are mean±SEM, n = 3. *<i>p</i><0.001 relative to Wt.</p