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Hemin is known to induce endocytosis of prion-protein (PrP(C)) from the neuronal plasma membrane, potentially limiting propagation of the disease causing PrP-scrapie (PrP(Sc)) isoform. Hemin is therefore an attractive disease-modifying option for sporadic Creutzfeldt-Jakob disease (sCJD), a human prion disorder with no effective treatment. The hemin-PrP(C) interaction is also of interest in cerebral-hemorrhage (CH), a condition where potentially toxic hemin molecules come in contact with neuronal PrP(C). Interestingly, PrP(C) is upregulated in penumbric neurons surrounding CH and is known to confer neuroprotection in a dose-dependent manner. The underlying mechanism, however, is not clear. Here, we report that hemin binds PrP(C) on diverse cell lines, resulting in its aggregation or degradation in a cell-type specific manner. Surprisingly, the hemin-PrP(C) interaction upregulates Hb synthesis in hematopoietic cells, a response reversed by deleting the hemin-binding octa-peptide repeat region of PrP(C). A similar response is noted in brain organotypic cultures where exposure to hemin induces significantly more α-globin in wild-type (PrP(+/+)) relative to PrP-knock-out (PrP(-/-)) samples. Furthermore, red blood cells and brain tissue from PrP(-/-) mice show significantly less α-globin relative to PrP(+/+) controls, indicating a positive effect of PrP(C) on Hb synthesis under physiological conditions as well. Surprisingly, levels of α-globin are significantly higher in sCJD brain tissue relative to controls, suggesting compensatory upregulation of Hb synthesis by surviving neurons or misregulation in diseased brains. These observations reveal a unique function of PrP(C) that is likely to impact the therapeutic management of CH and sCJD. [ABSTRACT FROM AUTHOR]

Abstract Iron is an essential biometal in the aqueous humor, the principal source of nutrients for the avascular cornea and the lens. Here, we explored whether the ciliary body (CB), the source of aqueous humor, transports iron, and if the prion protein (PrPC) facilitates this process as in the outer retina. Using a combination of human, bovine, and mouse eyes as models, we report the expression of iron export proteins ferroportin and ceruloplasmin, and major iron uptake and storage proteins transferrin, transferrin receptor, and ferritin in the ciliary epithelium, indicating active exchange of iron at this site. Ferroportin and transferrin receptor are also expressed in the corneal endothelium. However, the relative expression of iron export and uptake proteins suggests export from the ciliary epithelium and import by corneal endothelium. In addition, abundant expression of PrPC, a ferrireductase that facilitates iron transport, is noted in pigmented and non-pigmented epithelium of the CB, posterior pigmented epithelium of the iris, corneal endothelium and epithelium, and lens epithelium. Notably, majority of PrPC in the ciliary epithelium is cleaved at the β-site as in retinal pigment epithelial cells, suggesting a role in iron transport. Most of the PrPC in the cornea, however, is full-length, and susceptible to aggregation by intracerebrally inoculated PrP-scrapie, an infectious conformation of PrPC responsible for human and animal prion disorders. Soluble PrPC is present in the aqueous and vitreous humor, a provocative observation with significant implications. Together, these observations suggest independent cycling of iron in the anterior segment, and a prominent role of PrPC in this process. Aggregation of PrPC in the cornea of PrP-scrapie-infected animals raises the alarming possibility of transmission of animal prions through corneal abrasions. Highlights • Non-pigmented ciliary epithelium exports iron to the aqueous humor. • PrPC modulates iron transport across ciliary epithelium and corneal endothelium. • Soluble PrPC is present in the aqueous and vitreous humor. • Prion infection induces aggregation of PrPC in the cornea and ciliary epithelium. [ABSTRACT FROM AUTHOR]

Excess circulating iron is stored in the liver, and requires reduction of non-Tf-bound iron (NTBI) and transferrin (Tf) iron at the plasma membrane and endosomes, respectively, by ferrireductase (FR) proteins for transport across biological membranes through divalent metal transporters. Here, we report that prion protein (PrP C ), a ubiquitously expressed glycoprotein most abundant on neuronal cells, functions as a FR partner for divalent-metal transporter-1 (DMT1) and ZIP14. Thus, absence of PrP C in PrP-knock-out (PrP –/– ) mice resulted in markedly reduced liver iron stores, a deficiency that was not corrected by chronic or acute administration of iron by the oral or intraperitoneal routes. Likewise, preferential radiolabeling of circulating NTBI with 59 Fe revealed significantly reduced uptake and storage of NTBI by the liver of PrP –/– mice relative to matched PrP +/+ controls. However, uptake, storage, and utilization of ferritin-bound iron that does not require reduction for uptake were increased in PrP –/– mice, indicating a compensatory response to the iron deficiency. Expression of exogenous PrP C in HepG2 cells increased uptake and storage of ferric iron (Fe 3+ ), not ferrous iron (Fe 2+ ), from the medium, supporting the function of PrP C as a plasma membrane FR. Coexpression of PrP C with ZIP14 and DMT1 in HepG2 cells increased uptake of Fe 3+ significantly, and surprisingly, increased the ratio of N-terminally truncated PrP C forms lacking the FR domain relative to full-length PrP C . Together, these observations indicate that PrP C promotes, and possibly regulates, the uptake of NTBI through DMT1 and Zip14 via its FR activity. Implications of these observations for neuronal iron homeostasis under physiological and pathological conditions are discussed. [ABSTRACT FROM AUTHOR]

Brain iron-dyshomeostasis is an important cause of neurotoxicity in prion disorders, a group of neurodegenerative conditions associated with the conversion of prion protein (PrPC) from its normal conformation to an aggregated, PrP-scrapie (PrPSc) isoform. Alteration of iron homeostasis is believed to result from impaired function of PrPC in neuronal iron uptake via its ferrireductase activity. However, unequivocal evidence supporting the ferrireductase activity of PrPC is lacking. Kidney provides a relevant model for this evaluation because PrPC is expressed in the kidney, and ~370 µg of iron are reabsorbed daily from the glomerular filtrate by kidney proximal tubule cells (PT), requiring ferrireductase activity. Here, we report that PrPC promotes the uptake of transferrin (Tf) and non-Tf-bound iron (NTBI) by the kidney in vivo and mainly NTBI by PT cells in vitro. Thus, uptake of 59Fe administered by gastric gavage, intravenously, or intraperitoneally was significantly lower in PrPknock- out (PrP-/-) mouse kidney relative to PrP+/+ controls. Selective in vivo radiolabeling of plasma NTBI with 59Fe revealed similar results. Expression of exogenous PrPC in immortalized PT cells showed localization on the plasma membrane and intracellular vesicles and increased transepithelial transport of 59Fe-NTBI and to a smaller extent 59Fe-Tf from the apical to the basolateral domain. Notably, the ferrireductase-deficient mutant of PrP (PrPΔ51-89) lacked this activity. Furthermore, excess NTBI and hemin caused aggregation of PrPC to a detergent-insoluble form, limiting iron uptake. Together, these observations suggest that PrPC promotes retrieval of iron from the glomerular filtrate via its ferrireductase activity and modulates kidney iron metabolism. [ABSTRACT FROM AUTHOR]

Recently, we reported β-cleavage of the prion protein (PrPC) in human ocular tissues. Here, we explored whether this is unique to the human eye, and its functional implications. A comparison of the cleavage pattern of PrPC in human ocular tissues with common nocturnal and diurnal animals revealed mainly β-cleavage in humans, and mostly full-length PrPC in animal retinas. Soluble FL PrPC and N-terminal fragment (N2) released from β-cleavage was observed in the aqueous and vitreous humor (AH & VH). Expression of human PrPC in ARPE-19 cells, a human retinal pigmented epithelial cell line, also showed β-cleaved PrPC. Surprisingly, β-cleavage was not altered by a variety of insults, including oxidative stress, suggesting a unique role of this cleavage in the human eye. It is likely that β-cleaved C- or N-terminal fragments of PrPC protect from various insults unique to the human eye. On the contrary, β-cleaved C-terminus of PrPC is susceptible to conversion to the pathological PrP-scrapie form, and includes the binding sites for β1-integrin and amyloid-β, molecules implicated in several ocular disorders. Considering the species and tissue-specific cleavage of PrPC, our data suggest re-evaluation of prion infectivity and other ocular disorders of the human eye conducted in mouse models. • The prion protein (PrPC) is mainly β-cleaved in the human ocular tissue. • This contrasts with mainly α-cleaved PrPC in the human brain. • β-Cleaved PrPC is relatively more susceptible to prion infectivity. • This raises concerns of acquiring prion infection via the ocular route. • PrPC in most animals is α-cleaved, requiring re-evaluation of infectivity studies. [ABSTRACT FROM AUTHOR]

Prion protein (PrPC) is implicated in the pathogenesis of prion disorders, but its normal function is unclear. We demonstrate that PrPC is a ferrireductase (FR), and its absence causes systemic iron deficiency in PrP knock-out mice (PrP-/-). When exposed to non-transferrin-bound (NTB) radioactive-iron (59FeCl3) by gastric-gavage, PrP-/- mice absorb significantly more 59Fe from the intestinal lumen relative to controls, indicating appropriate systemic response to the iron deficiency. Chronic exposure to excess dietary iron corrects this deficiency, but unlike wild-type (PrP+/+) controls that remain iron over-loaded, PrP-/- mice revert back to the iron deficient phenotype after 5 months of chase on normal diet. Bone marrow (BM) preparations of PrP-/- mice on normal diet show relatively less stainable iron, and this phenotype is only partially corrected by intraperitoneal administration of excess iron-dextran. Cultured PrP-/- BM-macrophages incorporate significantly less NTB-59Fe in the absence or presence of excess extracellular iron, indicating reduced uptake and/or storage of available iron in the absence of PrPC. When expressed in neuroblastoma cells, PrPC exhibits NAD(P)H-dependent cell-surface and intracellular FR activity that requires the copper-binding octa-peptide-repeat region and linkage to the plasma membrane for optimal function. Incorporation of NTB-59Fe by neuroblastoma cells correlates with FR activity of PrPC, implicating PrPC in cellular iron uptake and metabolism. These observations explain the correlation between PrPC expression and cellular iron levels, and the cause of iron imbalance in sporadic-Creutzfeldt-Jakob-disease brains where PrPC accumulates as insoluble aggregates. [ABSTRACT FROM AUTHOR]

Abstract: Prion disease associated neurotoxicity is mainly attributed to PrP-scrapie (PrPSc), the disease associated isoform of a normal protein, the prion protein (PrPC). Participation of other proteins and processes is suspected, but their identity and contribution to the pathogenic process is unclear. Emerging evidence implicates imbalance of brain iron homeostasis as a significant cause of prion disease-associated neurotoxicity. The underlying cause of this change, however, remains unclear. We demonstrate that iron is sequestered in heat and SDS-stable protein complexes in sporadic-Creutzfeldt–Jakob-disease (sCJD) brains, creating a phenotype of iron deficiency. The underlying cause is change in the characteristics of ferritin, an iron storage protein that becomes aggregated, detergent-insoluble, and partitions with denatured ferritin using conventional methods of ferritin purification. A similar phenotype of iron deficiency is noted in the lumbar spinal cord (SC) tissue of scrapie infected hamsters, a site unlikely to be affected by massive neuronal death and non-specific iron deposition. As a result, the iron uptake protein transferrin (Tf) is upregulated in scrapie infected SC tissue, and increases with disease progression. A direct correlation between Tf and PrPSc suggests sequestration of iron in dysfunctional ferritin that either co-aggregates with PrPSc or is rendered dysfunctional by PrPSc through an indirect process. Surprisingly, amplification of PrPSc in vitro by the protein-misfolding-cyclic-amplification (PMCA) reaction using normal brain homogenate as substrate does not increase the heat and SDS-stable pool of iron even though both PrPSc and ferritin aggregate by this procedure. These observations highlight important differences between PrPSc-protein complexes generated in vivo during disease progression and in vitro by the PMCA reaction, and the significance of these complexes in PrPSc-associated neurotoxicity. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrPC) from a mainly a-helical to a b-sheet rich PrP-scrapie (PrPSc) form is the main event responsible for prion disease associated neurotoxicity. However, neither the mechanism of toxicity by PrPSc, nor the normal function of PrPC 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 PrPC 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 PrPC 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 PrPC on ferritin iron content is enhanced by stimulating PrPC endocytosis, and reversed by cross-linking PrPC on the plasma membrane. Expression of mutant PrP forms lacking the octapeptide-repeats, the membrane anchor, or carrying the pathogenic mutation PrP102L decreases ferritin iron content significantly relative to PrPC expressing cells, but the effect on cellular LIP and levels of Tf, TfR, and ferritin is complex, varying with the mutation. Neither PrPC nor the mutant PrP forms influence the rate or amount of iron released into the medium, suggesting a functional role for PrPC in cellular iron uptake and transport to ferritin, and dysfunction of PrPC as a significant contributing factor of brain iron imbalance in prion disorders. [ABSTRACT FROM AUTHOR]

Endothelial-to-mesenchyme-like transition (Endo-MT) of trabecular meshwork (TM) cells is known to be associated with primary open angle glaucoma (POAG). Here, we investigated whether the prion protein (PrPC), a neuronal protein known to modulate epithelial-to-mesenchymal transition in a variety of cell types, is expressed in the TM, and plays a similar role at this site. Using a combination of primary human TM cells and human, bovine, and PrP-knock-out (PrP−/−) mouse models, we demonstrate that PrPC is expressed in the TM of all three species, including endothelial cells lining the Schlemm's canal. Silencing of PrPC in primary human TM cells induces aggregation of β1-integrin and upregulation of α-smooth muscle actin, fibronectin, collagen 1A, vimentin, and laminin, suggestive of transition to a mesenchyme-like phenotype. Remarkably, intraocular pressure is significantly elevated in PrP−/− mice relative to wild-type controls, suggesting reduced pliability of the extracellular matrix and increased resistance to aqueous outflow in the absence of PrPC. Since PrPC is cleaved by members of the disintegrin and matrix-metalloprotease family that are increased in the aqueous humor of POAG arising from a variety of conditions, it is likely that concomitant cleavage of PrPC exaggerates and confounds the pathology by inducing Endo-MT-like changes in the TM. [ABSTRACT FROM AUTHOR]