Prion protein expression level alters regional copper, iron and zinc content in the mouse brain

The central role of the prion protein (PrP) in a family of fatal neurodegenerate diseases has garnered considerable research interest over the past two decades. Moreover, the role of PrP in neuronal development, as well as its apparent role in metal homeostasis, is increasingly of interest. The host-encoded form of the prion protein (PrP(C)) binds multiple copper atoms via its N-terminal domain and can influence brain copper and iron levels. The importance of PrP(C) to the regulation of brain metal homeostasis and metal distribution, however, is not fully understood. We therefore employed synchrotron-based X-ray fluorescence imaging to map the level and distributions of several key metals in the brains of mice that express different levels of PrP(C). Brain sections from wild-type, prion gene knockout (Prnp(-/-)) and PrP(C) over-expressing mice revealed striking variation in the levels of iron, copper, and even zinc in specific brain regions as a function of PrP(C) expression. Our results indicate that one important function of PrP(C) may be to regulate the amount and distribution of specific metals within the central nervous system. This raises the possibility that PrP(C) levels, or its activity, might regulate the progression of diseases in which altered metal homeostasis is thought to play a pathogenic role such as Alzheimer's, Parkinson's and Wilson's diseases and disorders such as hemochromatosis.     

The murine B cell repertoire is severely selected against endogenous cellular prion protein

Abs to the prion protein (PrP) can protect against experimental prion infections, but efficient Ab responses are difficult to generate because PrP is expressed on many tissues and induces a strong tolerance. We previously showed that immunization of wild-type mice with PrP peptides and CpG oligodeoxynucleic acid overcomes tolerance and induces cellular and humoral responses to PrP. In this study, we compared Ab and T cell repertoires directed to PrP in wild-type and PrP knockout (Prnp o/o) C57BL/6 mice. Animals were immunized with mouse PrP-plasmid DNA or with 30-mer overlapping peptides either emulsified in CFA or CpG/IFA. In Prnp o/o mice, Abs raised by PrP-plasmid DNA immunization recognized only N-terminal PrP peptides; analyses of Ab responses after PrP peptide/CFA immunization allowed us to identify six distinct epitopes, five of which were also recognized by Abs raised by PrP peptides/CpG. By contrast, in wild-type mice, no Ab response was detected after PrP-plasmid DNA or peptide/CFA immunization. However, when using CpG, four C-terminal peptides induced Abs specific for distinct epitopes. Importantly, immune sera from Prnp o/o but not from wild-type mice bound cell surface PrP. Abs of IgG1 and IgG2b subclasses predominated in Prnp o/o mice while the strongest signals were for IgG2b in wild-type mice. Most anti-PrP Th cells were directed to a single epitope in both Prnp o/o and wild-type mice. We conclude that endogenous PrPC expression profoundly affects the Ab repertoire as B cells reactive for epitopes exposed on native PrPC are strongly tolerized. Implications for immunotherapy against prion diseases are discussed.     

Impairment of superoxide dismutase activation by N-terminally truncated prion protein (PrP) in PrP-deficient neuronal cell line

Previous studies have reported a neuroprotective role for cellular prion protein (PrP(C)) against apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line, but the mechanisms remain unclear. In this study, to investigate the mechanisms by which PrP(C) prevents apoptosis, the authors compared apoptosis of Prnp(-/-) cells with that of Prnp(-/-) cells expressing the wild-type PrP(C) or PrP(C) lacking N-terminal octapeptide repeat region under serum-free conditions. Re-introduction of Prnp rescued cells from apoptosis, upregulated superoxide dismutase (SOD) activity, enhanced superoxide anion elimination, and inhibited caspase-3/9 activation. On the other hand, N-terminally truncated PrP(C) enhanced apoptosis accompanied by potentiation of superoxide production and caspase-3/9 activation due to inhibition of SOD. These results suggest that PrP(C) protects Prnp(-/-) cells from apoptosis via superoxide- and caspase-3/9-dependent pathways by upregulating SOD activity. Furthermore, the octapeptide repeat region of PrP(C) plays an essential role in regulating apoptosis and SOD activity.     

A naturally occurring C-terminal fragment of the prion protein (PrP) delays disease and acts as a dominant-negative inhibitor of PrPSc formation

The cellular prion protein (PrPC) undergoes constitutive proteolytic cleavage between residues 111/112 to yield a soluble N-terminal fragment (N1) and a membrane-anchored C-terminal fragment (C1). The C1 fragment represents the major proteolytic fragment of PrPC in brain and several cell types. To explore the role of C1 in prion disease, we generated Tg(C1) transgenic mice expressing this fragment (PrP(Δ23-111)) in the presence and absence of endogenous PrP. In contrast to several other N-terminally deleted forms of PrP, the C1 fragment does not cause a spontaneous neurological disease in the absence of endogenous PrP. Tg(C1) mice inoculated with scrapie prions remain healthy and do not accumulate protease-resistant PrP, demonstrating that C1 is not a substrate for conversion to PrPSc (the disease-associated isoform). Interestingly, Tg(C1) mice co-expressing C1 along with wild-type PrP (either endogenous or encoded by a second transgene) become ill after scrapie inoculation, but with a dramatically delayed time course compared with mice lacking C1. In addition, accumulation of PrPSc was markedly slowed in these animals. Similar effects were produced by a shorter C-terminal fragment of PrP(Δ23-134). These results demonstrate that C1 acts as dominant-negative inhibitor of PrPSc formation and accumulation of neurotoxic forms of PrP. Thus, C1, a naturally occurring fragment of PrPC, might play a modulatory role during the course of prion diseases. In addition, enhancing production of C1, or exogenously administering this fragment, represents a potential therapeutic strategy for the treatment of prion diseases.     

The N-terminal, polybasic region is critical for prion protein neuroprotective activity

Several lines of evidence suggest that the normal form of the prion protein, PrP(C), exerts a neuroprotective activity against cellular stress or toxicity. One of the clearest examples of such activity is the ability of wild-type PrP(C) to suppress the spontaneous neurodegenerative phenotype of transgenic mice expressing a deleted form of PrP (Δ32-134, called F35). To define domains of PrP involved in its neuroprotective activity, we have analyzed the ability of several deletion mutants of PrP (Δ23-31, Δ23-111, and Δ23-134) to rescue the phenotype of Tg(F35) mice. Surprisingly, all of these mutants displayed greatly diminished rescue activity, although Δ23-31 PrP partially suppressed neuronal loss when expressed at very high levels. Our results pinpoint the N-terminal, polybasic domain as a critical determinant of PrP(C) neuroprotective activity, and suggest that identification of molecules interacting with this region will provide important clues regarding the normal function of the protein. Small molecule ligands targeting this region may also represent useful therapeutic agents for treatment of prion diseases.     

Biological and biochemical characterization of mice expressing prion protein devoid of the octapeptide repeat region after infection with prions

Accumulating lines of evidence indicate that the N-terminal domain of prion protein (PrP) is involved in prion susceptibility in mice. In this study, to investigate the role of the octapeptide repeat (OR) region alone in the N-terminal domain for the susceptibility and pathogenesis of prion disease, we intracerebrally inoculated RML scrapie prions into tg(PrPΔOR)/Prnp(0/0) mice, which express mouse PrP missing only the OR region on the PrP-null background. Incubation times of these mice were not extended. Protease-resistant PrPΔOR, or PrP(Sc)ΔOR, was easily detectable but lower in the brains of these mice, compared to that in control wild-type mice. Consistently, prion titers were slightly lower and astrogliosis was milder in their brains. However, in their spinal cords, PrP(Sc)ΔOR and prion titers were abundant and astrogliosis was as strong as in control wild-type mice. These results indicate that the role of the OR region in prion susceptibility and pathogenesis of the disease is limited. We also found that the PrP(Sc)ΔOR, including the pre-OR residues 23-50, was unusually protease-resistant, indicating that deletion of the OR region could cause structural changes to the pre-OR region upon prion infection, leading to formation of a protease-resistant structure for the pre-OR region.     

Misfolding of the prion protein at the plasma membrane induces endocytosis, intracellular retention and degradation

Suramin induces misfolding of the cellular prion protein (PrP(C)) and interferes with the propagation of infectious scrapie prions. A mechanistic analysis of this effect revealed that suramin-induced misfolding occurs at the plasma membrane and is dependent on the proximal region of the C-terminal domain (aa 90-158) of PrP(C). The conformational transition induces rapid internalization, mediated by the unstructured N-terminal domain, and subsequent intracellular degradation of PrP(C). As a consequence, PrP Delta N adopts a misfolded conformation at the plasma membrane; however, internalization is significantly delayed. We also found that misfolding and intracellular retention of PrP(C) can be induced by copper and that, moreover, copper interferes with the propagation of the pathogenic prion protein (PrP(Sc)) in scrapie-infected N2a cells. Our study revealed a quality control pathway for aberrant PrP conformers present at the plasma membrane and identified distinct PrP domains involved.     

Prion protein (PrP) with amino-proximal deletions restoring susceptibility of PrP knockout mice to scrapie.

The 'protein only' hypothesis postulates that the prion, the agent causing transmissible spongiform encephalopathies, is PrP(Sc), an isoform of the host protein PrP(C). Protease treatment of prion preparations cleaves off approximately 60 N-terminal residues of PrP(Sc) but does not abrogate infectivity. Disruption of the PrP gene in the mouse abolishes susceptibility to scrapie and prion replication. We have introduced into PrP knockout mice transgenes encoding wild-type PrP or PrP lacking 26 or 49 amino-proximal amino acids which are protease susceptible in PrP(Sc). Inoculation with prions led to fatal disease, prion propagation and accumulation of PrP(Sc) in mice expressing both wild-type and truncated PrPs. Within the framework of the 'protein only' hypothesis, this means that the amino-proximal segment of PrP(C) is not required either for its susceptibility to conversion into the pathogenic, infectious form of PrP or for the generation of PrP(Sc).     

Expression of prion protein increases cellular copper binding and antioxidant enzyme activities but not copper delivery

The N-terminal region of the prion protein PrP(C) contains a series of octapeptide repeats. This region has been implicated in the binding of divalent metal ions, particularly copper. PrP(C) has been suggested to be involved in copper transport and metabolism and in cell defense mechanisms against oxidative insult, possibly through the regulation of the intracellular CuZn superoxide dismutase activity (CuZn-SOD) or a SOD-like activity of PrP(C) itself. However, up to now the link between PrP(C) expression and copper metabolism or SOD activity has still to be formally established; particularly because conflicting results have been obtained in vivo. In this study, we report a link between PrP(C), copper binding, and resistance to oxidative stress. Radioactive copper ((64)Cu) was used at a physiological concentration to demonstrate that binding of copper to the outer plasma cell membrane is related to the level of PrP(C) expression in a cell line expressing a doxycycline-inducible murine PrP(C) gene. Cellular PIPLC pretreatment indicated that PrP(C) was not involved in copper delivery at physiological concentrations. We also demonstrated that murine PrP(C) expression increases several antioxidant enzyme activities and glutathione levels. Prion protein may be a stress sensor sensitive to copper and able to initiate, following copper binding, a signal transduction process acting on the antioxidant systems to improve cell defenses.     

Protease-resistant prions selectively decrease Shadoo protein

The central event in prion diseases is the conformational conversion of the cellular prion protein (PrP(C)) into PrP(Sc), a partially protease-resistant and infectious conformer. However, the mechanism by which PrP(Sc) causes neuronal dysfunction remains poorly understood. Levels of Shadoo (Sho), a protein that resembles the flexibly disordered N-terminal domain of PrP(C), were found to be reduced in the brains of mice infected with the RML strain of prions [1], implying that Sho levels may reflect the presence of PrP(Sc) in the brain. To test this hypothesis, we examined levels of Sho during prion infection using a variety of experimental systems. Sho protein levels were decreased in the brains of mice, hamsters, voles, and sheep infected with different natural and experimental prion strains. Furthermore, Sho levels were decreased in the brains of prion-infected, transgenic mice overexpressing Sho and in infected neuroblastoma cells. Time-course experiments revealed that Sho levels were inversely proportional to levels of protease-resistant PrP(Sc). Membrane anchoring and the N-terminal domain of PrP both influenced the inverse relationship between Sho and PrP(Sc). Although increased Sho levels had no discernible effect on prion replication in mice, we conclude that Sho is the first non-PrP marker specific for prion disease. Additional studies using this paradigm may provide insight into the cellular pathways and systems subverted by PrP(Sc) during prion disease.     

Genetic mapping of activity determinants within cellular prion proteins: N-terminal modules in PrPC offset pro-apoptotic activity of the Doppel helix B/B' region

The PrP-like Doppel (Dpl) protein causes apoptotic death of cerebellar neurons in transgenic mice, a process prevented by expression of the wild type (wt) cellular prion protein, PrP(C). Internally deleted forms of PrP(C) resembling Dpl such as PrPDelta32-121 produce a similar PrP(C)-sensitive pro-apoptotic phenotype in transgenic mice. Here we demonstrate that these phenotypic attributes of wt Dpl, wt PrP(C), and PrPDelta132-121 can be accurately recapitulated by transfected mouse cerebellar granule cell cultures. This system was then explored by mutagenesis of the co-expressed prion proteins to reveal functional determinants. By this means, neuroprotective activity of wt PrP(C) was shown to be nullified by a deletion of the N-terminal charged region implicated in endocytosis and retrograde axonal transport (PrPDelta23-28), by deletion of all five octarepeats (PrPDelta51-90), or by glycine replacement of four octarepeat histidine residues required for selective binding of copper ions (Prnp"H/G"). In the case of Dpl, overlapping deletions defined a requirement for the gene interval encoding helices B and B' (DplDelta101-125). These data suggest contributions of copper binding and neuronal trafficking to wt PrP(C) function in vivo and place constraints upon current hypotheses to explain Dpl/PrP(C) antagonism by competitive ligand binding. Further implementation of this assay should provide a fuller understanding of the attributes and subcellular localizations required for activity of these enigmatic proteins.     

Effects of Copper on Survival of Prion Protein Knockout Neurons and Glia

Abstract: The N-terminal region of the prion protein (PrP) contains an octameric repeat region suggested to bind copper. A 32-amino acid peptide (PrPOcta) based on this region in the protein was tested for its effects on cultured cerebellar cells. Cerebellar cells from mice deficient in cellular PrP (Prnp^0/0 mice) are more sensitive to copper toxicity and oxidative stress. PrPOcta selectively promotes the survival of Prnp^0/0 cerebellar cells. However, PrPOcta also reduces the toxicity of CuSO₄ on cerebellar cells and abolishes the difference in increased sensitivity of Prnp^0/0 cells to both copper toxicity and also oxidative stress from xanthine oxidase. PrPOcta does not promote the survival or proliferation of astrocytes or microglia. The survival-promoting effects of PrPOcta on neurons may be due to its ability to effectively chelate copper. The octameric repeat region of PrP may represent a functional domain of the native protein.     

Stromal complement receptor CD21/35 facilitates lymphoid prion colonization and pathogenesis

We have studied the role of CD21/35, which bind derivatives of complement factors C3 and C4, in extraneural prion replication and neuroinvasion. Upon administration of small prion inocula, CD21/35(-/-) mice experienced lower attack rates and delayed disease over both wild-type (WT) mice and mice with combined C3 and C4 deficiencies. Early after inoculation, CD21/35(-/-) spleens were devoid of infectivity. Reciprocal adoptive bone marrow transfers between WT and CD21/35(-/-) mice revealed that protection from prion infection resulted from ablation of stromal, but not hemopoietic, CD21/35. Further adoptive transfer experiments between WT mice and mice devoid of both the cellular prion protein PrP(C) and CD21/35 showed that splenic retention of inoculum depended on stromal CD21/35 expression. Because both PrP(C) and CD21/35 are highly expressed on follicular dendritic cells, CD21/35 appears to be involved in targeting prions to follicular dendritic cells and expediting neuroinvasion following peripheral exposure to prions.     

The prion protein and lipid rafts

Prions are the causative agent of the transmissible spongiform encephalopathies, such as Creutzfeldt-Jakob disease in humans. In these prion diseases the normal cellular form of the prion protein (PrP(C)) undergoes a post-translational conformational conversion to the infectious form (PrP(Sc)). PrP(C) associates with cholesterol- and glycosphingolipid-rich lipid rafts through association of its glycosyl-phosphatidylinositol (GPI) anchor with saturated raft lipids and through interaction of its N-terminal region with an as yet unidentified raft associated molecule. PrP(C) resides in detergent-resistant domains that have different lipid and protein compositions to the domains occupied by another GPI-anchored protein, Thy-1. In some cells PrP(C) may endocytose through caveolae, but in neuronal cells, upon copper binding to the N-terminal octapeptide repeats, the protein translocates out of rafts into detergent-soluble regions of the plasma membrane prior to endocytosis through clathrin-coated pits. The current data suggest that the polybasic region at its N-terminus is required to engage PrP(C) with a transmembrane adaptor protein which in turn links with the clathrin endocytic machinery. PrP(C) associates in rafts with a variety of signalling molecules, including caveolin-1 and Fyn and Src tyrosine kinases. The clustering of PrP(C) triggers a range of signal transduction processes, including the recruitment of the neural cell adhesion molecule to rafts which in turn promotes neurite outgrowth. Lipid rafts appear to be involved in the conformational conversion of PrP(C) to PrP(Sc), possibly by providing a favourable environment for this process to occur and enabling disease progression.     

Copper converts the cellular prion protein into a protease-resistant species that is distinct from the scrapie isoform

Several lines of evidence have suggested that copper ions play a role in the biology of both PrP(C) and PrP(Sc), the normal and pathologic forms of the prion protein. To further investigate this intriguing connection, we have analyzed how copper ions affect the biochemical properties of PrP(C) extracted from the brains of transgenic mice and from transfected cells. We report that the metal rapidly and reversibly induces PrP(C) to become protease-resistant and detergent-insoluble. Although these two properties are commonly associated with PrP(Sc), we demonstrate using a conformation-dependent immunoassay that copper-treated PrP is structurally distinct from PrP(Sc). The effect of copper requires the presence of at least one of the five octapeptide repeats normally present in the N-terminal half of the protein, consistent with the idea that the metal alters the biochemical properties of PrP by directly binding to this region. These results suggest potential roles for copper in prion diseases, as well as in the physiological function of PrP(C).     

PrP cooperates with STI1 to regulate SOD activity in PrP-deficient neuronal cell line

Cellular prion protein (PrP(C)) plays anti-apoptotic and anti-oxidative roles in apoptosis induced by serum deprivation in an immortalized prion protein gene (Prnp)-deficient neuronal cell line. The octapeptide repeat region (OR) and N-terminal half of the hydrophobic region (HR) of PrP(C) are indispensable for PrP(C) activity, but the mechanisms remain unclear. In the present study, elucidation of the mechanisms by which PrP(C) elicits the anti-oxidative activities was facilitated by evidence of stress-inducible protein 1 (STI1) mediating PrP(C)-dependent superoxide dismutase (SOD) activation. Immunoprecipitation revealed that PrP(C) was associated with STI1. The inhibitory peptides against PrP(C)-STI1 binding [STI1 pep.1 and PrP(113-132)] indicated toxic activity in PrP(C)-expressing cells by inhibiting SOD activity but not in Prnp(-/-) cells. Furthermore, OR and N-terminal half of the HR were required for the inhibitory effect of PrP(113-132) but not STI1 pep.1. These data are consistent with results established with a model where OR and N-terminal half of the HR mediate the action of STI1 upon cell survival and upregulation of SOD activity.     

Differential contribution of superoxide dismutase activity by prion protein in vivo

Normal prion protein (PrP(C)) is a copper binding protein and may play a role in cellular resistance to oxidative stress. Recently, copper-bound recombinant PrP(C) has been shown to exhibit superoxide dismutase (SOD)-like activity. However, as PrP(C) affinity for copper is low in comparison to other cupro-proteins, the question remains as to whether PrP(C) could contribute SOD activity in vivo. To unravel this enigma, we compared the SOD activity in lysates extracted from different regions of the brain from wild-type mice before and after the depletion of PrP(C). We found that removal of PrP(C) from the brain lysates reduced the levels of total SOD activity. The level of contribution to the total SOD activity was correlated to the level of PrP expressed and to the predominant form of PrP present in the specific brain region. Collectively, these results provide strong evidence that PrP(C) differentially contributes to the total SOD activity in vivo.     

Comparative analysis of the human and chicken prion protein copper binding regions at pH 6.5

Recent experimental evidence supports the hypothesis that prion proteins (PrPs) are involved in the Cu(II) metabolism. Moreover, the copper binding region has been implicated in transmissible spongiform encephalopathies, which are caused by the infectious isoform of prion proteins (PrP(Sc)). In contrast to mammalian PrP, avian prion proteins have a considerably different N-terminal copper binding region and, most interestingly, are not able to undergo the conversion process into an infectious isoform. Therefore, we applied x-ray absorption spectroscopy to analyze in detail the Cu(II) geometry of selected synthetic human PrP Cu(II) octapeptide complexes in comparison with the corresponding chicken PrP hexapeptide complexes at pH 6.5, which mimics the conditions in the endocytic compartments of neuronal cells. Our results revealed that structure and coordination of the human PrP copper binding sites are highly conserved in the pH 6.5-7.4 range, indicating that the reported pH dependence of copper binding to PrP becomes significant at lower pH values. Furthermore, the different chicken PrP hexarepeat motifs display homologous Cu(II) coordination at sub-stoichiometric copper concentrations. Regarding the fully cation-saturated prion proteins, however, a reduced copper coordination capability is supposed for the chicken prion protein based on the observation that chicken PrP is not able to form an intra-repeat Cu(II) binding site. These results provide new insights into the prion protein structure-function relationship and the conversion process of PrP.