Abnormal Brain Iron Homeostasis in Human and Animal Prion Disorders

Neurotoxicity in all prion disorders is believed to result from the accumulation of PrP-scrapie (PrPSc), a β-sheet rich isoform of a normal cell-surface glycoprotein, the prion protein (PrPC). Limited reports suggest imbalance of brain iron homeostasis as a significant associated cause of neurotoxicity in prion-infected cell and mouse models. However, systematic studies on the generality of this phenomenon and the underlying mechanism(s) leading to iron dyshomeostasis in diseased brains are lacking. In this report, we demonstrate that prion disease–affected human, hamster, and mouse brains show increased total and redox-active Fe (II) iron, and a paradoxical increase in major iron uptake proteins transferrin (Tf) and transferrin receptor (TfR) at the end stage of disease. Furthermore, examination of scrapie-inoculated hamster brains at different timepoints following infection shows increased levels of Tf with time, suggesting increasing iron deficiency with disease progression. Sporadic Creutzfeldt-Jakob disease (sCJD)–affected human brains show a similar increase in total iron and a direct correlation between PrP and Tf levels, implicating PrPSc as the underlying cause of iron deficiency. Increased binding of Tf to the cerebellar Purkinje cell neurons of sCJD brains further indicates upregulation of TfR and a phenotype of neuronal iron deficiency in diseased brains despite increased iron levels. The likely cause of this phenotype is sequestration of iron in brain ferritin that becomes detergent-insoluble in PrPSc-infected cell lines and sCJD brain homogenates. These results suggest that sequestration of iron in PrPSc–ferritin complexes induces a state of iron bio-insufficiency in prion disease–affected brains, resulting in increased uptake and a state of iron dyshomeostasis. An additional unexpected observation is the resistance of Tf to digestion by proteinase-K, providing a reliable marker for iron levels in postmortem human brains. These data implicate redox-iron in prion disease–associated neurotoxicity, a novel observation with significant implications for prion disease pathogenesis

Prion Protein Modulates Cellular Iron Uptake: A Novel Function with Implications for Prion Disease Pathogenesis

Converging evidence leaves little doubt that a change in the conformation of prion protein (PrPC) from a mainly α-helical to a β-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

G Protein-Coupled Receptors Directly Bind Filamin A with High Affinity and Promote Filamin Phosphorylation

Although interaction of a few G protein-coupled receptors (GPCRs) with Filamin A, a key actin cross-linking and biomechanical signal transducer protein, has been observed, a comprehensive structure–function analysis of this interaction is lacking. Through a systematic sequence-based analysis, we found that a conserved filamin binding motif is present in the cytoplasmic domains of >20% of the 824 GPCRs encoded in the human genome. Direct high-affinity interaction of filamin binding motif peptides of select GPCRs with the Ig domain of Filamin A was confirmed by nuclear magnetic resonance spectroscopy and isothermal titration calorimetric experiments. Engagement of the filamin binding motif with the Filamin A Ig domain induced the phosphorylation of filamin by protein kinase A in vitro. In transfected cells, agonist activation as well as constitutive activation of representative GPCRs dramatically elicited recruitment and phosphorylation of cellular Filamin A, a phenomenon long known to be crucial for regulating the structure and dynamics of the cytoskeleton. Our data suggest a molecular mechanism for direct GPCR–cytoskeleton coupling via filamin. Until now, GPCR signaling to the cytoskeleton was predominantly thought to be indirect, through canonical G protein-mediated signaling cascades involving GTPases, adenylyl cyclases, phospholipases, ion channels, and protein kinases. We propose that the GPCR-induced filamin phosphorylation pathway is a conserved, novel biochemical signaling paradigm

Scrapie-infected mouse brains show alteration of iron homeostasis.

<p>(A) Normal (NH) and scrapie-infected (MoSc) mouse brains collected at end stage of disease were homogenized and fractionated by SDS-PAGE, followed by immunoblotting. Probing with specific antibodies shows increased expression of PrP, ferritin, and Tf in MoSc samples compared to matched controls (lanes 1–7). (B) Quantitative analysis after normalization with β-actin shows a significant increase in PrP, ferritin, and Tf expression in diseased samples compared to matched controls. Values are mean±SEM. *<i>p</i><0.0001, **<i>p</i><0.001 relative to NH samples. n = 7. (C) Dot blot analysis as in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000336#ppat-1000336-g001" target="_blank">Figure 1C</a> shows a prominent reaction for Fe (II) and Fe (III) iron in MoSc homogenates compared to matched controls.</p

Correlation and linear reggression analysis of human autopsy samples.

<p>(A) Correlation between PrP and different proteins shows a strong positive correlation between PrP and Tf, PrP and iron, and Tf and iron in CJD+ cases. CJD− cases show no correlation between PrP and Tf and between iron and Tf, and modest correlation between PrP and iron. (B) Quantification of total iron shows two distinct groups of CJD− cases with high and low iron content, the higher values arising most likely from specific disease conditions (see <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1000336#s4" target="_blank">Materials and Methods</a>). In contrast, CJD+ cases show consistent iron levels in all 20 cases that are significantly higher than the apparently “disease-free” control group. Values are mean±SEM. *<i>p</i><0.001. n = 20. (C) Linear regression analysis shows a positive correlation between PrP and Tf in CJD+ cases (R linear = 0.444 for CJD+, and 0.05 for CJD−). n = 20.</p

Scrapie-infected cells show co-localization of PrP and ferritin.

<p>ScN2a and SMB cells cultured in the absence or presence of 0.1 mM FAC were immunostained with 8H4-anti-mouse-FITC followed by anti-ferritin–anti-rabbit-TRITC. Untreated ScN2a cells show minimal PrP reactivity and reaction for ferritin in the cytosol (panels 1–3). Following exposure to FAC, both PrP and ferritin form intracellular aggregates that co-localize for the most part (panels 4–6, arrowheads). Untreated SMB cells show intracellular aggregates of PrP and reaction for ferritin in the cytosol (panels 7–9). However, exposure to FAC increases the reactivity for PrP and ferritin significantly, and, notably, PrP and ferritin form intracellular aggregates that co-localize in vesicular structures (panels 10–12, arrowheads).</p