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In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution

View Article: PubMed Central - PubMed

ABSTRACT

Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein- mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein- mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments.

No MeSH data available.


Comparison of Cu, Fe, and metal-binding protein expression levels in wild-type and PrPC- mouse spleen at different ages. (A) The graph shows the ratio of Cu and Fe levels in Prnp0/0 and Prnp+/+ spleen samples (P15 N = 3; P30 N = 4; P90, P180 N = 6; P365 N = 5). (B) The graph shows the weight of spleen extracted from Prnp0/0 and Prnp+/+ mice; N = 4. (C) Representative Western blot images showing metal-binding protein levels in Prnp0/0 and Prnp+/+ spleen samples. The constant level of the housekeeping protein (β-Actin) are also reported. (D) The graph shows the up- or down-regulation of protein expression in Prnp0/0 samples compared to Prnp+/+, i.e., (Prnp0/0 protein OD/housekeeping OD)/ (Prnp+/+ protein OD/housekeeping OD); N = 4. All error bars indicate SD; *p < 0.05; **p < 0.01; ***p < 0.001.
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Figure 3: Comparison of Cu, Fe, and metal-binding protein expression levels in wild-type and PrPC- mouse spleen at different ages. (A) The graph shows the ratio of Cu and Fe levels in Prnp0/0 and Prnp+/+ spleen samples (P15 N = 3; P30 N = 4; P90, P180 N = 6; P365 N = 5). (B) The graph shows the weight of spleen extracted from Prnp0/0 and Prnp+/+ mice; N = 4. (C) Representative Western blot images showing metal-binding protein levels in Prnp0/0 and Prnp+/+ spleen samples. The constant level of the housekeeping protein (β-Actin) are also reported. (D) The graph shows the up- or down-regulation of protein expression in Prnp0/0 samples compared to Prnp+/+, i.e., (Prnp0/0 protein OD/housekeeping OD)/ (Prnp+/+ protein OD/housekeeping OD); N = 4. All error bars indicate SD; *p < 0.05; **p < 0.01; ***p < 0.001.

Mentions: Taking into account the alterations observed in the liver of Prnp0/0 mice for iron and copper metabolism, we investigated the effects of PrPC absence on spleen, another fundamental organ for metal ion homeostasis. First, we confirmed the presence of PrPC in the spleen (Figure S3). Then, we measured copper and iron content in wild-type and PrPC- mouse spleen at the different ages, and expressed results as the ratio between Prnp0/0 and Prnp+/+ ion concentration values (Figure 3A). Results expressed in μg/mL are reported in Figures S1F,G. Conversely from the liver, we detected a strong reduction in copper content and an increase in iron level starting from P30 in Prnp0/0 spleen (Figure 3A). The copper content reduction is likely related to alterations in its uptake due to PrPC ablation and lower Steap3 expression detected in PrPC- spleen (Figures 3C,D). Steap3 is indeed involved in reducing Cu2+ to Cu+ for subsequent internalization via Ctr1. Reduced Steap3 level suggests an impairment of copper uptake leading to copper deficiency in PrPC- mouse spleen. Expression levels of the copper-binding proteins Cp and Atp7b revealed no differences between Prnp+/+ and Prnp0/0 mouse spleen (Figure S5A), suggesting the correct incorporation of copper into Cp. Indeed, oxidase activity in spleen is not altered in PrPC- mice, as shown in Figure S5B.


In Absence of the Cellular Prion Protein, Alterations in Copper Metabolism and Copper-Dependent Oxidase Activity Affect Iron Distribution
Comparison of Cu, Fe, and metal-binding protein expression levels in wild-type and PrPC- mouse spleen at different ages. (A) The graph shows the ratio of Cu and Fe levels in Prnp0/0 and Prnp+/+ spleen samples (P15 N = 3; P30 N = 4; P90, P180 N = 6; P365 N = 5). (B) The graph shows the weight of spleen extracted from Prnp0/0 and Prnp+/+ mice; N = 4. (C) Representative Western blot images showing metal-binding protein levels in Prnp0/0 and Prnp+/+ spleen samples. The constant level of the housekeeping protein (β-Actin) are also reported. (D) The graph shows the up- or down-regulation of protein expression in Prnp0/0 samples compared to Prnp+/+, i.e., (Prnp0/0 protein OD/housekeeping OD)/ (Prnp+/+ protein OD/housekeeping OD); N = 4. All error bars indicate SD; *p < 0.05; **p < 0.01; ***p < 0.001.
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Figure 3: Comparison of Cu, Fe, and metal-binding protein expression levels in wild-type and PrPC- mouse spleen at different ages. (A) The graph shows the ratio of Cu and Fe levels in Prnp0/0 and Prnp+/+ spleen samples (P15 N = 3; P30 N = 4; P90, P180 N = 6; P365 N = 5). (B) The graph shows the weight of spleen extracted from Prnp0/0 and Prnp+/+ mice; N = 4. (C) Representative Western blot images showing metal-binding protein levels in Prnp0/0 and Prnp+/+ spleen samples. The constant level of the housekeeping protein (β-Actin) are also reported. (D) The graph shows the up- or down-regulation of protein expression in Prnp0/0 samples compared to Prnp+/+, i.e., (Prnp0/0 protein OD/housekeeping OD)/ (Prnp+/+ protein OD/housekeeping OD); N = 4. All error bars indicate SD; *p < 0.05; **p < 0.01; ***p < 0.001.
Mentions: Taking into account the alterations observed in the liver of Prnp0/0 mice for iron and copper metabolism, we investigated the effects of PrPC absence on spleen, another fundamental organ for metal ion homeostasis. First, we confirmed the presence of PrPC in the spleen (Figure S3). Then, we measured copper and iron content in wild-type and PrPC- mouse spleen at the different ages, and expressed results as the ratio between Prnp0/0 and Prnp+/+ ion concentration values (Figure 3A). Results expressed in μg/mL are reported in Figures S1F,G. Conversely from the liver, we detected a strong reduction in copper content and an increase in iron level starting from P30 in Prnp0/0 spleen (Figure 3A). The copper content reduction is likely related to alterations in its uptake due to PrPC ablation and lower Steap3 expression detected in PrPC- spleen (Figures 3C,D). Steap3 is indeed involved in reducing Cu2+ to Cu+ for subsequent internalization via Ctr1. Reduced Steap3 level suggests an impairment of copper uptake leading to copper deficiency in PrPC- mouse spleen. Expression levels of the copper-binding proteins Cp and Atp7b revealed no differences between Prnp+/+ and Prnp0/0 mouse spleen (Figure S5A), suggesting the correct incorporation of copper into Cp. Indeed, oxidase activity in spleen is not altered in PrPC- mice, as shown in Figure S5B.

View Article: PubMed Central - PubMed

ABSTRACT

Essential elements as copper and iron modulate a wide range of physiological functions. Their metabolism is strictly regulated by cellular pathways, since dysregulation of metal homeostasis is responsible for many detrimental effects. Neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and prion diseases are characterized by alterations of metal ions. These neurodegenerative maladies involve proteins that bind metals and mediate their metabolism through not well-defined mechanisms. Prion protein, for instance, interacts with divalent cations via multiple metal-binding sites and it modulates several metal-dependent physiological functions, such as S-nitrosylation of NMDA receptors. In this work we focused on the effect of prion protein absence on copper and iron metabolism during development and adulthood. In particular, we investigated copper and iron functional values in serum and several organs such as liver, spleen, total brain and isolated hippocampus. Our results show that iron content is diminished in prion protein- mouse serum, while it accumulates in liver and spleen. Our data suggest that these alterations can be due to impairments in copper-dependent cerulopalsmin activity which is known to affect iron mobilization. In prion protein- mouse total brain and hippocampus, metal ion content shows a fluctuating trend, suggesting the presence of homeostatic compensatory mechanisms. However, copper and iron functional values are likely altered also in these two organs, as indicated by the modulation of metal-binding protein expression levels. Altogether, these results reveal that the absence of the cellular prion protein impairs copper metabolism and copper-dependent oxidase activity, with ensuing alteration of iron mobilization from cellular storage compartments.

No MeSH data available.