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Prion protein modulates cellular iron uptake: a novel function with implications for prion disease pathogenesis.

Singh A, Mohan ML, Isaac AO, Luo X, Petrak J, Vyoral D, Singh N - PLoS ONE (2009)

Bottom Line: As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased.The positive effect of PrP(C) on ferritin iron content is enhanced by stimulating PrP(C) endocytosis, and reversed by cross-linking PrP(C) on the plasma membrane.Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP(102L) decreases ferritin iron content significantly relative to PrP(C) expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation.

View Article: PubMed Central - PubMed

Affiliation: The Department of Pathology, Case Western Reserve University, Cleveland, OH, USA.

ABSTRACT
Converging evidence leaves little doubt that a change in the conformation of prion protein (PrP(C)) from a mainly alpha-helical to a beta-sheet rich PrP-scrapie (PrP(Sc)) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrP(Sc), nor the normal function of PrP(C) is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrP(C) mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrP(C) increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrP(C) on ferritin iron content is enhanced by stimulating PrP(C) endocytosis, and reversed by cross-linking PrP(C) on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP(102L) decreases ferritin iron content significantly relative to PrP(C) expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrP(C) nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrP(C) in cellular iron uptake and transport to ferritin, and dysfunction of PrP(C) as a significant contributing factor of brain iron imbalance in prion disorders.

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PrP is not involved in the export of iron from cells.(A) Cells expressing PrPC, PrPΔ51–89, PrPΔ23–89, and PrP102L were radiolabeled with 59FeCl3, washed with PBS supplemented with DFO, and chased in complete medium for 30, 60, 90, 120 min, and 16 hours. At the indicated time points equal aliquots of medium samples were quantified in a γ-counter. Estimation of released 59Fe does not show a significant difference between the indicated cell lines at any time point. n = 6 experiments in triplicate. (B) Cell associated 59Fe after 16 h of chase reflects the ferritin iron content of each cell line noted in Figure 1 above, though the difference between cell lines is significantly less. (C) Possible ferroxidase activity of recombinant PrP was measured using the established colorimetric method [44] with modifications. Negative controls included water and albumin supplemented with copper, and positive controls included plasma in the absence or presence of copper. Recombinant PrP does not show detectable ferroxidase activity either in the absence or presence of copper, whereas plasma shows a robust reaction under similar conditions.
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pone-0004468-g009: PrP is not involved in the export of iron from cells.(A) Cells expressing PrPC, PrPΔ51–89, PrPΔ23–89, and PrP102L were radiolabeled with 59FeCl3, washed with PBS supplemented with DFO, and chased in complete medium for 30, 60, 90, 120 min, and 16 hours. At the indicated time points equal aliquots of medium samples were quantified in a γ-counter. Estimation of released 59Fe does not show a significant difference between the indicated cell lines at any time point. n = 6 experiments in triplicate. (B) Cell associated 59Fe after 16 h of chase reflects the ferritin iron content of each cell line noted in Figure 1 above, though the difference between cell lines is significantly less. (C) Possible ferroxidase activity of recombinant PrP was measured using the established colorimetric method [44] with modifications. Negative controls included water and albumin supplemented with copper, and positive controls included plasma in the absence or presence of copper. Recombinant PrP does not show detectable ferroxidase activity either in the absence or presence of copper, whereas plasma shows a robust reaction under similar conditions.

Mentions: To determine if the difference in cellular iron levels between cell lines is due to differential release into the medium, M17, PrPC, PrPΔ51–89, PrPΔ23–89, and PrP102L cells were cultured in the presence of 3H-thymidine overnight to monitor cell proliferation and radiolabeled with 59FeCl3 for 4 hours as above. Labeled cells were washed with PBS containing 100 µM DFO to remove surface bound 59Fe, and chased in complete medium for 30 minutes to 16 hours. At the indicated times equal aliquots of medium were retrieved and released 59Fe was quantified in a γ-counter. Kinetic analysis shows minimal difference in extracellular iron between cell lines after normalizing with 3H-thymidine (Fig. 9A). Estimation of cell-associated 59Fe after 16 hours of chase shows more 59Fe in PrPC and PrP102L, and significantly less in PrPΔ51–89 and PrPΔ23–89 compared to M17 lysates as observed in Fig. 1 above (Fig. 9B). However, the fold difference in ferritin iron content between M17 and other cell lines is significantly less after 16 hours of chase, and represents steady state levels of iron content in each cell line. Evaluation of possible ferroxidase activity of recombinant PrP using plasma as a positive control yielded negative results (Fig. 9C). Though informative, this result does not rule out possible ferroxidase activity of cell-associated PrP, a technically challenging assay that has yielded inconclusive results (data not shown).


Prion protein modulates cellular iron uptake: a novel function with implications for prion disease pathogenesis.

Singh A, Mohan ML, Isaac AO, Luo X, Petrak J, Vyoral D, Singh N - PLoS ONE (2009)

PrP is not involved in the export of iron from cells.(A) Cells expressing PrPC, PrPΔ51–89, PrPΔ23–89, and PrP102L were radiolabeled with 59FeCl3, washed with PBS supplemented with DFO, and chased in complete medium for 30, 60, 90, 120 min, and 16 hours. At the indicated time points equal aliquots of medium samples were quantified in a γ-counter. Estimation of released 59Fe does not show a significant difference between the indicated cell lines at any time point. n = 6 experiments in triplicate. (B) Cell associated 59Fe after 16 h of chase reflects the ferritin iron content of each cell line noted in Figure 1 above, though the difference between cell lines is significantly less. (C) Possible ferroxidase activity of recombinant PrP was measured using the established colorimetric method [44] with modifications. Negative controls included water and albumin supplemented with copper, and positive controls included plasma in the absence or presence of copper. Recombinant PrP does not show detectable ferroxidase activity either in the absence or presence of copper, whereas plasma shows a robust reaction under similar conditions.
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Related In: Results  -  Collection

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pone-0004468-g009: PrP is not involved in the export of iron from cells.(A) Cells expressing PrPC, PrPΔ51–89, PrPΔ23–89, and PrP102L were radiolabeled with 59FeCl3, washed with PBS supplemented with DFO, and chased in complete medium for 30, 60, 90, 120 min, and 16 hours. At the indicated time points equal aliquots of medium samples were quantified in a γ-counter. Estimation of released 59Fe does not show a significant difference between the indicated cell lines at any time point. n = 6 experiments in triplicate. (B) Cell associated 59Fe after 16 h of chase reflects the ferritin iron content of each cell line noted in Figure 1 above, though the difference between cell lines is significantly less. (C) Possible ferroxidase activity of recombinant PrP was measured using the established colorimetric method [44] with modifications. Negative controls included water and albumin supplemented with copper, and positive controls included plasma in the absence or presence of copper. Recombinant PrP does not show detectable ferroxidase activity either in the absence or presence of copper, whereas plasma shows a robust reaction under similar conditions.
Mentions: To determine if the difference in cellular iron levels between cell lines is due to differential release into the medium, M17, PrPC, PrPΔ51–89, PrPΔ23–89, and PrP102L cells were cultured in the presence of 3H-thymidine overnight to monitor cell proliferation and radiolabeled with 59FeCl3 for 4 hours as above. Labeled cells were washed with PBS containing 100 µM DFO to remove surface bound 59Fe, and chased in complete medium for 30 minutes to 16 hours. At the indicated times equal aliquots of medium were retrieved and released 59Fe was quantified in a γ-counter. Kinetic analysis shows minimal difference in extracellular iron between cell lines after normalizing with 3H-thymidine (Fig. 9A). Estimation of cell-associated 59Fe after 16 hours of chase shows more 59Fe in PrPC and PrP102L, and significantly less in PrPΔ51–89 and PrPΔ23–89 compared to M17 lysates as observed in Fig. 1 above (Fig. 9B). However, the fold difference in ferritin iron content between M17 and other cell lines is significantly less after 16 hours of chase, and represents steady state levels of iron content in each cell line. Evaluation of possible ferroxidase activity of recombinant PrP using plasma as a positive control yielded negative results (Fig. 9C). Though informative, this result does not rule out possible ferroxidase activity of cell-associated PrP, a technically challenging assay that has yielded inconclusive results (data not shown).

Bottom Line: As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased.The positive effect of PrP(C) on ferritin iron content is enhanced by stimulating PrP(C) endocytosis, and reversed by cross-linking PrP(C) on the plasma membrane.Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP(102L) decreases ferritin iron content significantly relative to PrP(C) expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation.

View Article: PubMed Central - PubMed

Affiliation: The Department of Pathology, Case Western Reserve University, Cleveland, OH, USA.

ABSTRACT
Converging evidence leaves little doubt that a change in the conformation of prion protein (PrP(C)) from a mainly alpha-helical to a beta-sheet rich PrP-scrapie (PrP(Sc)) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrP(Sc), nor the normal function of PrP(C) is entirely clear. Recent reports suggest that imbalance of iron homeostasis is a common feature of prion infected cells and mouse models, implicating redox-iron in prion disease pathogenesis. In this report, we provide evidence that PrP(C) mediates cellular iron uptake and transport, and mutant PrP forms alter cellular iron levels differentially. Using human neuroblastoma cells as models, we demonstrate that over-expression of PrP(C) increases intra-cellular iron relative to non-transfected controls as indicated by an increase in total cellular iron, the cellular labile iron pool (LIP), and iron content of ferritin. As a result, the levels of iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) are decreased, and expression of iron storage protein ferritin is increased. The positive effect of PrP(C) on ferritin iron content is enhanced by stimulating PrP(C) endocytosis, and reversed by cross-linking PrP(C) on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP(102L) decreases ferritin iron content significantly relative to PrP(C) expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrP(C) nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrP(C) in cellular iron uptake and transport to ferritin, and dysfunction of PrP(C) as a significant contributing factor of brain iron imbalance in prion disorders.

Show MeSH
Related in: MedlinePlus