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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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Endocytosis of PrP increases ferritin iron content.(A) PrPC-cells exposed to 1 µg/ml of 3F4 for 5 days were radiolabeled with 59FeCl3 for 4 hours, and lysates were fractionated on a non-denaturing gel and auto-radiographed (lanes 1 and 2). Equal aliquots of the same samples were boiled in SDS-containing sample buffer and fractionated in duplicate by SDS-PAGE followed by immunoblotting with PrP specific antibodies 3F4 and 8H4 (lanes 3–6). Subsequently, the membranes were re-probed for ferritin, Tf, TfR, and β-actin (lanes 7 and 8). (B) Quantification by densitometry shows an increase in ferritin iron and TfR levels, and insignificant change in Tf levels in 3F4 exposed cells. Values are mean±SEM of three independent experiments. *p<0.001 compared to untreated cells. (C) Estimation of LIP after exposing the cells to 1 µg/ml of 3F4 or anti-Thy-1 antibody for 5 days shows insignificant difference between untreated and 3F4 treated PrPC cells, and a decrease in anti-Thy-1 treated cells. *p<0.001. n = 5.
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pone-0004468-g007: Endocytosis of PrP increases ferritin iron content.(A) PrPC-cells exposed to 1 µg/ml of 3F4 for 5 days were radiolabeled with 59FeCl3 for 4 hours, and lysates were fractionated on a non-denaturing gel and auto-radiographed (lanes 1 and 2). Equal aliquots of the same samples were boiled in SDS-containing sample buffer and fractionated in duplicate by SDS-PAGE followed by immunoblotting with PrP specific antibodies 3F4 and 8H4 (lanes 3–6). Subsequently, the membranes were re-probed for ferritin, Tf, TfR, and β-actin (lanes 7 and 8). (B) Quantification by densitometry shows an increase in ferritin iron and TfR levels, and insignificant change in Tf levels in 3F4 exposed cells. Values are mean±SEM of three independent experiments. *p<0.001 compared to untreated cells. (C) Estimation of LIP after exposing the cells to 1 µg/ml of 3F4 or anti-Thy-1 antibody for 5 days shows insignificant difference between untreated and 3F4 treated PrPC cells, and a decrease in anti-Thy-1 treated cells. *p<0.001. n = 5.

Mentions: The effect of increased endocytosis of PrPC on ferritin iron content was evaluated by radiolabeling cells cultured in the presence of 1 µg/ml of 3F4 with 59FeCl3 for the last 4 hours of the incubation, and analyzing radiolabeled lysates as in Figure 1 above. Fractionation by non-denaturing page shows a significant increase in ferritin iron in the 3F4 exposed lysate compared to untreated control (Fig. 7A, lanes 1 and 2, open arrow). Analysis by SDS-PAGE and immunoblotting shows 2–3 fold increase in reactivity for all PrP glycoforms with anti-PrP antibodies 3F4 and 8H4 (Fig. 7A, lanes 3–6). However, the 18 kDa fragment that results from recycling of PrPC from the plasma membrane is not increased in 3F4 exposed lysates, indicating stimulation of PrPC internalization and possible intracellular accumulation by 3F4 binding rather than increased recycling from the plasma membrane (Fig. 7A, lanes 5 and 6) [28]. The 50 kDa band represents internalized 3F4 (Fig. 7A, lanes 4 and 6). Immunobloting for ferritin, Tf, and TfR shows an increase in TfR, and minimal change in ferritin and Tf levels (Fig. 7A, lanes 7 and 8). Quantification by densitometry shows an increase in ferritin iron to 271%, and insignificant change in ferritin and Tf levels by 3F4 treatment. The increase in TfR levels to 175% is probably due to co-endocytosis with PrP-antibody complex (Fig. 7B). Measurement of cellular LIP revealed insignificant difference between 3F4 exposed and untreated controls after 24 hours (data not shown) or 5 days of treatment, indicating efficient transport of iron to ferritin within this time frame (Fig. 7C). PrPC cells treated with anti-Thy-1 antibody, however, demonstrated a significant decrease in LIP after 5 days of incubation with 3F4 (Fig. 7C).


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)

Endocytosis of PrP increases ferritin iron content.(A) PrPC-cells exposed to 1 µg/ml of 3F4 for 5 days were radiolabeled with 59FeCl3 for 4 hours, and lysates were fractionated on a non-denaturing gel and auto-radiographed (lanes 1 and 2). Equal aliquots of the same samples were boiled in SDS-containing sample buffer and fractionated in duplicate by SDS-PAGE followed by immunoblotting with PrP specific antibodies 3F4 and 8H4 (lanes 3–6). Subsequently, the membranes were re-probed for ferritin, Tf, TfR, and β-actin (lanes 7 and 8). (B) Quantification by densitometry shows an increase in ferritin iron and TfR levels, and insignificant change in Tf levels in 3F4 exposed cells. Values are mean±SEM of three independent experiments. *p<0.001 compared to untreated cells. (C) Estimation of LIP after exposing the cells to 1 µg/ml of 3F4 or anti-Thy-1 antibody for 5 days shows insignificant difference between untreated and 3F4 treated PrPC cells, and a decrease in anti-Thy-1 treated cells. *p<0.001. n = 5.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2637434&req=5

pone-0004468-g007: Endocytosis of PrP increases ferritin iron content.(A) PrPC-cells exposed to 1 µg/ml of 3F4 for 5 days were radiolabeled with 59FeCl3 for 4 hours, and lysates were fractionated on a non-denaturing gel and auto-radiographed (lanes 1 and 2). Equal aliquots of the same samples were boiled in SDS-containing sample buffer and fractionated in duplicate by SDS-PAGE followed by immunoblotting with PrP specific antibodies 3F4 and 8H4 (lanes 3–6). Subsequently, the membranes were re-probed for ferritin, Tf, TfR, and β-actin (lanes 7 and 8). (B) Quantification by densitometry shows an increase in ferritin iron and TfR levels, and insignificant change in Tf levels in 3F4 exposed cells. Values are mean±SEM of three independent experiments. *p<0.001 compared to untreated cells. (C) Estimation of LIP after exposing the cells to 1 µg/ml of 3F4 or anti-Thy-1 antibody for 5 days shows insignificant difference between untreated and 3F4 treated PrPC cells, and a decrease in anti-Thy-1 treated cells. *p<0.001. n = 5.
Mentions: The effect of increased endocytosis of PrPC on ferritin iron content was evaluated by radiolabeling cells cultured in the presence of 1 µg/ml of 3F4 with 59FeCl3 for the last 4 hours of the incubation, and analyzing radiolabeled lysates as in Figure 1 above. Fractionation by non-denaturing page shows a significant increase in ferritin iron in the 3F4 exposed lysate compared to untreated control (Fig. 7A, lanes 1 and 2, open arrow). Analysis by SDS-PAGE and immunoblotting shows 2–3 fold increase in reactivity for all PrP glycoforms with anti-PrP antibodies 3F4 and 8H4 (Fig. 7A, lanes 3–6). However, the 18 kDa fragment that results from recycling of PrPC from the plasma membrane is not increased in 3F4 exposed lysates, indicating stimulation of PrPC internalization and possible intracellular accumulation by 3F4 binding rather than increased recycling from the plasma membrane (Fig. 7A, lanes 5 and 6) [28]. The 50 kDa band represents internalized 3F4 (Fig. 7A, lanes 4 and 6). Immunobloting for ferritin, Tf, and TfR shows an increase in TfR, and minimal change in ferritin and Tf levels (Fig. 7A, lanes 7 and 8). Quantification by densitometry shows an increase in ferritin iron to 271%, and insignificant change in ferritin and Tf levels by 3F4 treatment. The increase in TfR levels to 175% is probably due to co-endocytosis with PrP-antibody complex (Fig. 7B). Measurement of cellular LIP revealed insignificant difference between 3F4 exposed and untreated controls after 24 hours (data not shown) or 5 days of treatment, indicating efficient transport of iron to ferritin within this time frame (Fig. 7C). PrPC cells treated with anti-Thy-1 antibody, however, demonstrated a significant decrease in LIP after 5 days of incubation with 3F4 (Fig. 7C).

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