I

Octapepiide repeats T N-linked glycan

Disulfide bond I Hydrophobic region Gp| anchQr

Fig. 1. Schematic structure of mammalian PrPC. The likely positions of the copper binding sites are indicated.

of brain- and cell-derived protein (37). Copper also causes aggregation of a synthetic peptide derived from residues 106-126 of PrP, thereby enhancing its toxicity to cultured neurons (40). Finally, copper has been found to produce covalent modifications of PrPC. The metal causes oxidation of recombinant PrP, involving either methionine or histidine residues (41,42) and also induces a cleavage of the N-terminus of the protein in the presence of hydrogen peroxide (43).

4.3. Enzymatic Activity

Several recent studies have suggested that PrPC may function as a cuproenzyme. Recombinant PrP refolded in the presence of copper has been reported to exhibit a superoxide dismutase (SOD) activity, as did PrPC immunoprecipitated from brain tissue (34,44-47). This enzymatic activity depended on the presence of the octapeptide repeats as well as on bound copper ions and was not affected by KCN, thus distinguishing it from the activity of Cu-Zn SOD. In addition, immunodepletion of PrPC from brain extracts was found to cause a reduction in SOD activity (48).

Although it would be certainly intriguing if PrPC functioned as an SOD-like enzyme, the biological significance of these results is uncertain for several reasons. First, the enzymatic activity measured for recombinant PrP depended on refolding the protein from a denatured state in the presence of 5 mM copper, which is a highly supraphysiological concentration and is inconsistent with the ability of micromolar concentrations of the metal to bind to the octapeptide repeats of the protein after it has already been folded. Second, even small organic molecules like amino acids can bind copper and exhibit weak dismutase activity, calling into question whether the protein moiety contributes at all to the SOD activity measured for PrPC. Finally, copper binds much more weakly to PrPC than to known cuproenzymes like Cu-Zn SOD, which must be denatured to remove bound metal, arguing against the possibility that the copper plays a specific catalytic role in PrPC.

Whether PrPC displays other copper-dependent enzymatic activities is unclear. There are two reports that the octapeptide repeat region of PrP can function as a copper reductase, an activity that depended on the presence of tryptophan residues (49,50). In contrast, another study indicated that rather than being subject to reduction, Cu(II) bound to the octapeptide repeats is maintained in a redox-inactive state (51). These reports have yet to be followed up.

Fig. 2. Cu2+ causes PrP to become proteinase K resistant. Detergent extracts of mouse brain were incubated with the indicated concentrations of CuSO4 for 30 min and then digested with different amounts of proteinase K (PK) for 30 min at 37°C. After termination of the digestion with PMSF, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with either anti-PrP antibody or with anti-actin antibody. The lanes containing undigested samples (0 |lg/mL PK) represent 8 |lg of protein, and the other lanes represent 40 |lg of protein. (Reprinted from ref. 39.)

Fig. 2. Cu2+ causes PrP to become proteinase K resistant. Detergent extracts of mouse brain were incubated with the indicated concentrations of CuSO4 for 30 min and then digested with different amounts of proteinase K (PK) for 30 min at 37°C. After termination of the digestion with PMSF, proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotted with either anti-PrP antibody or with anti-actin antibody. The lanes containing undigested samples (0 |lg/mL PK) represent 8 |lg of protein, and the other lanes represent 40 |lg of protein. (Reprinted from ref. 39.)

4.4. Cellular Trafficking

Our previous studies revealed that PrPC constitutively cycles between the plasma membrane and an early endocytic compartment in neuronal cells, with clathrin-coated pits mediating endocytic uptake of the protein (52,53). We have found that copper causes a dramatic alteration in this cellular trafficking pathway (54; L. R. Brown and D. A. Harris, unpublished). Initially, we observed that incubation of cells with Cu2+ concentrations above 100 |M for 30-90 min caused a marked reduction in the total amount of PrPC on the cell surface, as determined by biotinylation or by immunofluores-cence staining. This effect was also seen with Zn2+, but not with Co2+, Mn2+, Cd2+, Ni2+, or Fe2+. The effect, which was observed for mammalian as well as chicken PrPC expressed in neuroblastoma cells, was temperature dependent (it did not occur at 4°C) and was rapidly reversible (within minutes). Neither copper nor zinc had any effect on the distribution of the transferrin receptor, suggesting that the metals were not causing a generalized stimulation of endocytosis.

Several lines of evidence indicated that the primary effect of copper is to stimulate the endocytosis of PrPC, with relatively little change in the rate of recycling. To measure endocytosis and recycling, we used biochemical methods to follow the internalization of surface PrPC molecules that had been labeled by iodination or biotinylation (54). Alternatively, we used immunocytochemical techniques to visualize the metal-induced redistribution of surface PrPC molecules that had been prelabeled with anti-PrP antibodies (L. R. Brown and D. A. Harris, unpublished). We found that the antibody-tagged protein was translocated from the cell surface to punctate intracellular compartments in the presence of copper (Fig. 3). The internalized PrPC partially colocalized with both fluorescent transferrin and fluorescent wheat germ agglutinin, but not with LysoTracker (a fluorescent lysosomotropic amine), implying that the protein was being delivered to early endosomes and the Golgi, but not to lysosomes.

Copper-induced endocytosis of PrPC depends on the presence of the histidine-containing repeats, implying that the effect is the result of binding of the metal to PrPC rather to some other cellular protein that indirectly modulates endocytosis. Chicken or mouse PrPC molecules in which the repeats have been deleted or the critical histidine residues mutated are poorly endocytosed in response to copper (54,55). Interestingly, an insertionally mutated form of PrP containing 14 octapeptide repeats

Fig. 3. Copper causes endocytosis of antibody-labeled PrPC. N2a cells expressing chicken PrP were labeled with PrP antibody at 4°C (left panel) and were then warmed to 37°C for 30 min either in the absence of metal (middle panel) or in the presence of 250 |J,M CuSO4 (right panel).

that is associated with familial Creutzfeldt-Jakob disease is also refractory to copper-induced endocytosis (55), implying that the normal complement of five repeats is necessary for optimal copper binding or for whatever structural change the metal induces that triggers endocytosis. We have hypothesized that copper binding may enhance the affinity of PrPC for a putative receptor on the cell surface that is required for targeting to clathrin-coated pits (54).

4.5. Copper Content of Cells and Tissues

An initial report indicated that the content of copper, but not of several other transition metals, is only 10% of normal in crude membranes, synaptosomes, and endosomes derived from the brains of Prn-pm mice, which carry a disrupted PrP gene (23). Moreover, removal of surface PrPC from wildtype cerebellar neurons using a phospholipase dramatically reduced the membrane copper content. Copper content was determined in these experiments using X-ray fluorescence and atomic absorption spectroscopy. A subsequent study from the same authors (11) reported that synaptosomes from Prn-p010 mice had a copper content that was 50% of the wild-type level, a considerably smaller difference than in the original report. Based on these results, the authors proposed that PrPC may play a role in regulating copper release at the synapse (56).

We have re-examined this subject, using mass spectrometry to measure the concentrations of several transition metals in brain tissue from wild-type and Prn-p010 mice, as well as in Tga20 mice that overexpress PrP by 10-fold. We were unable to find any differences in metal content in either whole brain or of several subcellular fractions among mice of these three genotypes (57) and we believe that the results of Brown and colleagues (11,23) are likely to be in error.

4.6. Oxidative Stress

A number of pieces of evidence suggest that PrPC may play a role in protecting cells from oxida-tive stress and this function has been proposed to involve the ability of the protein to bind copper (reviewed in ref. 56). The most direct evidence for a protective role of PrPC is the observation that neurons cultured from the brains of Prn-p010 mice are more susceptible to oxidative stress induced by several different agents, including xanthine oxidase, copper, and hydrogen peroxide (58-60). Conversely, PC12 cells selected for resistance to oxidative stress (or resistance to copper toxicity) have higher levels of PrPC (61). In addition, several studies suggest that protein and lipid markers of oxidative stress are increased in brain tissue from Prn-p010 mice (62,63).

How might PrPC contribute to protection from oxidative stress? Certainly, one possible explanation would be that the protein itself possesses a copper-dependent SOD activity, which could protect cells from superoxide radicals generated in the extracellular space, by analogy to the role of cytoplas-mic Cu-Zn SOD. As discussed earlier, however, we feel that the reported SOD-like activity of copper-refolded PrPC is unlikely to be physiologically significant. Another suggested hypothesis is that PrPC is important for the delivery of copper ions to Cu-Zn SOD. It has been reported that the enzymatic activity and the 64Cu loading of Cu-Zn SOD from the brains of Prn-p0/0 mice is 10-50% of normal (59,62,64). Conversely, it has been claimed that the activity and copper loading of Cu-Zn SOD are increased in PrP-overexpressing mice. However, as shown in Fig. 4, we have been unable to replicate these two sets of results (57). The activities of other antioxidant enzymes such as catalase and glutathione reductase have been reported to be decreased in Prn-p0/0 mice (60,62), but whether PrPC plays a direct role in regulating these molecules remains to be determined. At this point, then, the mechanism by which PrPC protects cells from oxidative stress and whether this process involves binding of copper ions remain uncertain.

5. COPPER AND PrPSc

There are now several results that suggest a connection between copper and PrPSc, the pathogenic isoform of PrP. First, copper facilitates restoration of protease resistance and infectivity during refolding of guanidine-denatured PrPSc (65). Second, the protease cleavage pattern of PrPSc derived from the brains of CJD patients is altered by addition of metal-chelating agents and by the readdition of copper and zinc in micromolar concentrations (66). In fact, the cleavage patterns of two different PrPSc subtypes, corresponding to distinct prion strains, can be interconverted by manipulation of their bound metal. These effects of copper may not be the result of direct binding of the metal to PrPSc, because it has been reported that, in contrast to PrPC, PrPSc does not bind to a copper-loaded affinity column (67). In fact, it is possible that loss of metal binding by PrP could play a role in prion-induced pathology. Finally, it was reported almost 25 yr ago that administration of the copper-chelat-ing agent cuprizone to mice caused a spongiform degeneration of the brain similar to scrapie (68). Altogether, these results suggest that alterations in copper metabolism could play some role in the pathogenesis of prion diseases and even raise the possibility that manipulation of copper levels could represent a therapeutic modality.

6. CONCLUSIONS

A considerable body of data now indicates some connection between PrP and copper ions. How is one to evaluate this evidence, some of which has proven to be controversial, and what is its biological significance? First, we will address the possible role of copper in the physiological function of PrPC. Probably the most compelling and widely agreed upon observation is that copper ions bind to PrPC. It is clear that the histidine-containing octapeptide repeats represent one set of binding sites and that additional binding sites in more C-terminal locations also exist. Although there has been debate about the absolute value of the binding constants, it seems clear that PrPC binds copper with an affinity that is considerably lower than that of bona fide cuproenzymes like Cu-Zn SOD and ceruloplasmin, which need to be denatured to remove their bound metal. Thus, it is unlikely that copper serves an enzymatic function in PrPC, such as catalyzing the dismutation of superoxide anions. Rather, the affinity of PrPC for copper is similar to that of amino acids, peptides, and proteins like albumin in the extracellular medium. Thus, it seems most likely that the role of PrPC involves the reversible binding of copper ions that have been transferred from other extracellular ligands. Consistent with this idea, the total concentration of Cu2+ in plasma and cerebrospinal fluid (1-10 |M) is similar to the estimated Kd for copper binding to PrPC and the concentration of the metal in brain tissue is estimated to be even higher (100 |M) (69).

Our observation that copper stimulates the endocytosis of PrPCsuggests the hypothesis that PrPC functions as a recycling receptor for the cellular uptake or efflux of copper ions. In an uptake model, PrPC on the plasma membrane binds Cu2+ via the peptide repeats and then delivers the metal by endocytosis to an acidic, endosomal compartment. Copper ions then dissociate from PrPC by virtue of the

Fig. 4. Cu-Zn SOD protein, activity, and copper incorporation are similar in cultures of cerebellar neurons from Prn-p0/0, wild-type, and Tga20 mice. Lysates of cerebellar cultures were subjected to electrophoresis on a 10% polyacrylamide gel under nondenaturing conditions. (A) Western blot analysis of lysates using an antiserum against Cu-Zn SOD; (B) gel-based assay for SOD activity was performed using nitro blue tetrazolium; (C) autoradiography of lysates from 64Cu-labeled cultures. (Reprinted from ref. 57.)

Fig. 4. Cu-Zn SOD protein, activity, and copper incorporation are similar in cultures of cerebellar neurons from Prn-p0/0, wild-type, and Tga20 mice. Lysates of cerebellar cultures were subjected to electrophoresis on a 10% polyacrylamide gel under nondenaturing conditions. (A) Western blot analysis of lysates using an antiserum against Cu-Zn SOD; (B) gel-based assay for SOD activity was performed using nitro blue tetrazolium; (C) autoradiography of lysates from 64Cu-labeled cultures. (Reprinted from ref. 57.)

low endosomal pH and, after reduction to Cu1+, are transported into the cytoplasm by a transmembrane transporter. PrPC subsequently returns to the cell surface to bind additional copper, and the cycle is repeated. This proposed function for PrPC is analogous to that of the transferrin receptor in uptake of iron, with the exception that metal ions bind directly to the receptor in the case of PrPC rather than to a protein carrier comparable to transferrin. In a second model, PrPC serves as a receptor that facilitates cellular efflux of copper via the secretory pathway. PrPC is first delivered via endosomal vesicles to the trans-Golgi network or other post-Golgi compartments, and it then serves to bind copper ions that have been pumped into these compartments during transit to the cell surface in secretory vesicles. In addition to acting as a carrier for copper ions, PrPC could also play a role in specifically transferring the metal from the Menkes or Wilson transporters to secreted cuproproteins such as ceruloplas-min by physically interacting with these molecules. Our immunocytochemical localization of copper-internalized PrPC in both endosomes and the Golgi is consistent with either an uptake or efflux model.

It remains to be proven whether these models, or other ones, will turn out to be correct. The fact that, in our hands, the copper content of brain fractions from Prn-p0/0 mice is normal (57) would seem to indicate that if PrPC is involved in cellular uptake or efflux of copper, it is not likely to represent the primary or major pathway. Rather, PrPC may be part of a more specialized copper trafficking pathway. This conclusion is also consistent with our observation (at variance with ref. 70) that cells expressing different amounts of PrPC do not show obvious differences in net uptake of 64Cu (Pauly and Harris, unpublished data). In addition, the fact that Prn-p010 mice do not, in our experiments, have reduced Cu-Zn SOD or cytochrome oxidase levels (57) suggests that PrPC is not involved in the specialized pathways involved in copper delivery to these two cuproenzymes. Because several pieces of evidence indicate that neurons from Prn-p010 mice are more susceptible to oxidative stress, it is possible that PrPC-mediated copper uptake plays a role in delivery of the metal to other enzymes capable of protecting cells from oxidative damage.

Finally, it is possible that copper plays some role in prion diseases. Alterations in metal metabolism are known to be involved in the pathogenesis of several other neurodegenerative disorders (71). Although the primary pathology in prion diseases is likely to be the result of a toxic effect of PrPSc or some other abnormal form of PrP (77), it is possible that loss of a copper-related function of PrPC contributes in an ancillary way to the disease phenotype (e.g., via oxidative damage or abnormalities in metal trafficking) (72-74). In addition, copper may play a role in the conversion of PrPC to PrPSc. The fact that copper-treated PrP is protease resistant but reactive with 3F4 monoclonal antibody raises the possibility that this form of the protein represents a physical state that is intermediate between that of PrPC and PrPSc. Thus, some additional biochemical alteration might convert copper-bound PrP fully and irreversibly to the scrapie form. Intermediate states of PrP have been postulated on the basis of theoretical considerations (75), and PrP species that are distinct from both PrPC and PrPSc have been postulated to be the primary neurotoxic species in some prion diseases (76,77). It is thus possible to envisage that copper either initiates or modulates the production of pathogenic PrP molecules in prion diseases and that manipulation of copper levels may represent a strategy for treating these disorders.

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