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Human prion diseases are characterized by the conversion of the normal prion protein (PrPC) into a pathogenic isomer (PrPSc). Distinct PrPSc conformers are associated with different subtypes of prion diseases. PrPC binds copper and has antioxidation activity. Changes in metal-ion occupancy can lead to significant decline of the antioxidation activity and changes in conformation of the protein. We studied the trace element status of brains from patients with sporadic Creutzfeldt–Jakob disease (sCJD). We found a decrease of up to 50% of copper and an increase in manganese of approximately 10-fold in the brain tissues from sCJD subjects. We have also studied the metal occupancy of PrP in sCJD patients. We observed striking elevation of manganese and, to a lesser extent, of zinc accompanied by significant reduction of copper bound to purified PrP in all sCJD variants, determined by the PrP genotype and PrPSc type, combined. Both zinc and manganese were undetectable in PrPC preparations from controls. Copper and manganese changes were pronounced in sCJD subjects homozygous for methionine at codon 129 and carrying PrPSc type-1. Anti-oxidation activity of purified PrP was dramatically reduced by up to 85% in the sCJD variants, and correlated with increased in oxidative stress markers in sCJD brains. These results suggest that altered metal-ion occupancy of PrP plays a pivotal role in the pathogenesis of prion diseases. Since the metal changes differed in each sCJD variants, they may contribute to the diversity of PrPSc and disease phenotype in sCJD. Finally, this study also presented two potential approaches in the diagnosis of CJD; the significant increase in brain manganese makes it potentially detectable by MRI, and the binding of manganese by PrP in sCJD might represent a novel diagnostic marker.
The central event in the pathogenesis of prion diseases, which include Creutzfeldt–Jakob disease (CJD) in humans and scrapie in animals, is believed to be the post-translational conversion of the normal cellular prion protein (PrPC) into an abnormal isoform called scrapie PrP (PrPSc) that is partially resistant to proteases and is associated with transmissible disease (Prusiner 1998). Recent studies have showed that PrPC not only binds copper (Cu) within the octarepeat region located in the unstructured N-terminus (Brown et al. 1997), but under certain specific circumstances may bind along the C-terminal structured domain of protein fragments (Cereghetti et al. 2001). Furthermore, recombinant PrPC can also bind other metal ions such as manganese (Mn) (Brown et al. 2000) at both the octarepeats and the C-terminal sites (Collinge 2001). However, when Mn replaces Cu, PrP reportedly changes conformation and loses function (Brown et al. 2000). Indeed, accumulating evidence suggests that metallochemical alterations may play a role in the pathogenesis of prion diseases and other neurodegenerative diseases (Bush 2000).
Increasing evidence indicates that the phenotypic diversity of prion diseases is related to the multiple conformations that PrPSc may adopt (Monari et al. 1994; Parchi et al. 2000). Two major, but not exclusive, determinants of the disease phenotype in sporadic CJD (sCJD) are thought to be the genotype at codon 129, the site of a common methionine (M)/valine (V) polymorphism (Goldfarb et al. 1992), and the PrPSc type as determined by the size of the protease-resistant PrPSc, which in turn, is an indication of the PrPSc conformation (Monari et al. 1994; Parchi et al. 1999, 2000). Two main types and several minor types of PrPSc have been observed in CJD (Parchi et al. 1999, 2000). PrPSc type-1 and type-2 results from the cleavage of PrPSc by proteinase K (PK) at residues 82 and 97, respectively, while secondary cleavages around the two main cleavage sites generate the minor PrPSc types (Parchi et al. 2000). It has been proposed that in sCJD, the genotype at codon 129 affects the conformation of PrPSc and thus the site of protease cleavage (Parchi et al. 2000). In turn, PrPSc species with different conformations have been associated with distinct disease phenotypes (Monari et al. 1994; Telling et al. 1996; Parchi et al. 2000). Indirect evidence suggests that the level of metal-ion occupancy in PrP might also affect the conformation of PrPSc associated with human prion diseases (Wadsworth et al. 1999).
It has been demonstrated that both recombinant and brain-derived PrP have superoxide dismutase (SOD)-like activity when Cu is bound to the octarepeat region resulting in conformational changes to the protein (Brown et al. 1999, 2001; Wong et al. 2000b). When Cu is replaced with Mn in recombinant PrP, it loses the SOD-like activity (Brown et al. 2000). At the same time, there is increasing evidence linking oxidative stress to several neurodegenerative diseases (Bush 2000), including animal prion disease (Guentchev et al. 2000). All these motivate us to investigate (i) whether, like in Alzheimer's disease (Deibel et al. 1996), metal binding is altered in sCJD, the most common human prion disease, (ii) whether these alterations correlate with disease phenotype such as PrPSc type and PrP genotype at codon 129, and (iii) whether metal imbalances also correlate with PrP loses antioxidant function. These studies were carried out in brain tissues and affinity purified PrP preparations (i.e. PrPC, PrPSc and possibly other abnormal PrP species) obtained from four major variants of sCJD identified according to the genotype at codon 129 of the PrP gene and the PrPSc type as established by Parchi et al. (1999). We report that Cu binding to PrP purified from sCJD was significantly decreased while the binding of Mn and Zn was markedly increased. The diminution of bound Cu was especially severe in PrP preparations containing PrPSc type-1, while bound Mn showed a more pronounced increase in PrP preparations from sCJD subjects homozygous for methionine (MM) at codon 129. SOD-like activity was reduced by approximately 85% in each of the sCJD variants examined.
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- Materials and methods
Recent advances add new complexity to the pathogenesis of prion diseases. As originally proposed, the prion hypothesis postulates that a change in conformation leads to the conversion of PrPC to PrPSc, which then causes the disease (Prusiner 1998). Now it appears that metal-ions such as Cu and Mn, may contribute to the pathogenesis of prion diseases. It is well established that PrPC binds Cu (Brown et al. 1997; Cereghetti et al. 2001), but recent data suggest that PrPC binding of Cu is needed to induce conformational changes (Brown et al. 1999, 2001; Wong et al. 2000b) that enable PrPC to function as an antioxidant (Brown et al. 1999). On the other hand, the replacement of Cu by other metals such as Mn, not only causes the loss of this antioxidant activity but also increases PrP β-sheet content and resistance to proteases suggesting that these aberrations in metal binding change the conformation of PrP (Brown et al. 2000). According to Wadsworth et al. (1999) the conformation of some of the PrPSc species associated with specific CJD phenotypes could be affected by the level of metal binding in vitro. Therefore, these results collectively argue that abnormal metal binding by PrP may promote PrPSc formation. Furthermore, the level of metal occupancy might contribute to distinct conformations of PrPSc and the expression of different disease phenotypes. Since these altered PrP species lack SOD-like activity they are likely to expose brain cells to oxidative stress facilitating neurodegeneration.
Previous studies on the effects of metal binding on PrPC or PrPSc-like species have been carried out either in vitro (Brown et al. 2000) or are based on an indirect approach (Wadsworth et al. 1999). In this report, we directly measured and compared with controls the quantities of Cu, Mn and Zn cations as well as the SOD-like activities that are present in brain tissues or are directly associated with affinity purified PrP (PrPC, PrPSc and possibly other abnormal PrP species) obtained from human subjects affected by the major phenotypic variants of sCJD. We found a striking decrease of bound Cu and quantities of Mn cations exceeding those of Cu in the purified PrP preparations from sCJD subjects as compared with controls, while Mn cations were undetectable in PrPC preparations from controls. Similarly, Zn cations were detectable only on PrP preparations isolated from sCJD subjects. These changes were also accompanied by striking alterations in the brain metals concentrations; a significant decrease of Cu cations and a dramatic increase of Mn cations in brain tissues from sCJD subjects. These metal binding aberrations were associated with an ∼ 85% diminution of the SOD-like activity of PrP present in sCJD. The present data suggest that in sCJD, Mn and perhaps Zn replace Cu in binding to the total PrP population (PrPC, PrPSc and related abnormal PrP species), and that this replacement leads to the loss of the SOD-like activity associated with PrPC found in non-CJD. They also support the suggestion that metal binding aberrations may play a role in the pathogenesis of prion diseases by causing the loss of the antioxidation function associated with PrPC. The sizable increase of markers associated with oxidative damage, such as protein oxidation and lipid peroxidation end products, also argue for a failure of antioxidation activities in sCJD. Currently, oxidative stress is thought to be a pivotal event in several neurodegenerative diseases although it is likely that the mechanisms leading to it are different in the various diseases (Bush 2000; Butterfield and Kanski 2001). Indeed, to our knowledge the elevation in Mn that we showed to be so prominent in brain tissue and PrP preparations from subjects with sCJD has not been reported in other neurodegenerative diseases. Alterations in brain Cu and Zn (Deibel et al. 1996) but not Mn (Markesbery et al. 1984) have been observed in Alzheimer's disease. Moreover, the level of Mn does not change significantly during aging (Markesbery et al. 1984).
The alterations in metal binding we observed were not uniformly distributed among the different phenotypic variants of sCJD as determined by the genotype at codon 129 of the PrP gene and the PrPSc type. In brain tissue, the changes in Cu and Mn cations were more prominent in the variants associated with MM homozygosity than with VV homozygous variants while no definite difference was detected according to the PrPSc type. In contrast, Cu was decreased significantly more in purified preparations containing PrPSc type-1 than in those with PrPSc type-2. However, Mn increase was more prominent in MM homozygous cases. Combined, our data show that MM homozygosity at codon 129 and the presence of type-1 PrPSc are the two conditions associated with the most pronounced changes in Cu and Mn. Accordingly, the sCJD variant MM type-1, which corresponds to the classical and by far the most common CJD subtype (Parchi et al. 1999), has the most prominent Cu and Mn variations. Zn cations appear to distribute differently, since in brain tissue they are significantly decreased in cases carrying PrPSc type-1 and increased in the PrPSc type-2 cases, while in PrP preparations they are increased in all preparations but significantly more in PrPSc type-2 than type-1 preparations.
Wadsworth et al. (1999) reported that the gel migration characteristics of certain PrPSc species, hence their conformation, depend on their Cu and Zn ion occupancy. This was based on the observation that chemically chelating for these two metal-ions affected the conformation of PrPSc, hence their susceptibility toward proteinase K digestion. Although it is not easy to compare our data with those by Wadsworth et al. (1999) because of differences in our classification of human CJD (Parchi et al. 1997), data from both groups collectively point to variations in metal-ions occupancy as a possible determinant of PrP and phenotypic diversities in prion diseases.
In conclusion, we have shown that major alterations in the distribution and PrP association of metal-ions occur in sCJD; these changes may play a pivotal role in the pathogenesis of prion diseases as they lead to the loss of antioxidant function associated with PrPC and may promote the diversity of PrPSc and other abnormal PrP species. Whether the metal imbalance is a contributory cause or a mere consequence of PrPC conversion to abnormal isoforms remains to be determined. Finally, this study also presented two potential approaches in the diagnosis of human CJD; the significant increase of Mn in the frontal cortex makes it potentially detectable by clinical magnetic resonance imaging because of its paramagnetic property, and the binding of Mn by PrP in sCJD, but not in the control, might represent a novel diagnostic marker.