Interaction of metals with prion protein: Possible role of divalent cations in the pathogenesis of prion diseases

NeuroToxicology 27 (2006) 777–787

Interaction of metals with prion protein: Possible role of divalent cations in the pathogenesis of prion diseases Christopher J. Choi, Arthi Kanthasamy, Vellareddy Anantharam, Anumantha G. Kanthasamy * Parkinson’s Disorder Research Laboratory, Iowa Center for Advanced Neurotoxicology, Department of Biomedical Sciences, Iowa State University, 2062 Veterinary Medicine Building, Ames, IA 50011-1250, USA Received 15 January 2006; accepted 3 June 2006 Available online 18 June 2006

Abstract Prion diseases are fatal neurodegenerative disorders that affect both humans and animals. The rapid clinical progression, change in protein conformation, cross-species transmission and massive neuronal degeneration are some key features of this devastating degenerative condition. Although the etiology is unknown, aberrant processing of cellular prion proteins is well established in the pathogenesis of prion diseases. Normal cellular prion protein (PrPc) is highly conserved in mammals and expressed predominantly in the brain. Nevertheless, the exact function of the normal prion protein in the CNS has not been fully elucidated. Prion proteins may function as a metal binding protein because divalent cations such as copper, zinc and manganese can bind to octapeptide repeat sequences in the N-terminus of PrPc. Since the binding of these metals to the octapeptide has been proposed to influence both structural and functional properties of prion proteins, alterations in transition metal levels can alter the course of the disease. Furthermore, cellular antioxidant capacity is significantly compromised due to conversion of the normal prion protein (PrPc) to an abnormal scrapie prion (PrPsc) protein, suggesting that oxidative stress may play a role in the neurodegenerative process of prion diseases. The combination of imbalances in cellular transition metals and increased oxidative stress could further exacerbate the neurotoxic effect of PrPsc. This review includes an overview of the structure and function of prion proteins, followed by the role of metals such as copper, manganese and iron in the physiological function of the PrPc, and the possible role of transition metals in the pathogenesis of the prion disease. # 2006 Elsevier Inc. All rights reserved. Keywords: Transmissible spongiform encephalopathy; Scrapie; PrP; Neurotoxicity; Copper; Manganese; Iron; Metals

1. Introduction Prion diseases refer to a class of fatal transmissible spongiform encephalopathy (TSE) that affects the central nervous system (CNS) of both humans and animals. The well-known prion diseases include bovine spongiform encephalopathy (BSE) in cows, chronic wasting disease (CWD) in deer, scrapie in sheep, and Creutzfeldt-Jakob Disease (CJD) in humans (Collinge and Palmer, 1992). Table 1 summarizes various forms of prion diseases that affect both humans and animals. Once thought to be caused by a virulent particle, the cause of the disease was later proven to be due to an abnormal conformation of a host protein (Chatigny and Prusiner, 1980). Studies have shown that the propagation of the infectious prion protein cannot occur in the

* Corresponding author. Tel.: +1 515 294 2516; fax: +1 515 294 2315. E-mail address: [email protected] (A.G. Kanthasamy). 0161-813X/$ – see front matter # 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.neuro.2006.06.004

absence of host prion proteins (Prusiner et al., 1993), indicating the causal role of prion proteins in the pathogenesis. Prion diseases cause severe neuronal damage mainly in the brain regions that coordinate motor function, including the basal ganglia, cerebral cortex, thalamus, and cerebellum (Baringer et al., 1983; Gibbs et al., 1985; Hainfellner et al., 1997). The major neurological symptoms are extrapyramidal motor signs, cerebellar ataxia, pyramidal signs, and myoclonus (Brown and Gajdusek, 1991; Haltia et al., 1991). The neuronal damage is characterized pathologically as massive neuronal degeneration associated with accumulation of the abnormal prion protein PrPsc (scrapie isoform) derived from the normal prion protein PrPc (cellular isoform) through unknown cellular mechanisms (Collinge, 2001; Ma et al., 2002). The neuropathological changes include vacuolation of neutrophils, neuronal loss, and gliosis in the brain of diseased animals and humans (Owen et al., 1989; Collinge and Palmer, 1992; Palmer and Collinge, 1992). Incubation time of the disease can range anywhere from a few

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Table 1 Various forms of prion diseases in humans and animals Mechanism of pathogenesis Human prion disease Sporadic

Spontaneous conversion of PrPc to PrPsc? (unknown)

sCJD sFI Familial fCJD GSS fFI Infectious vCJD iCJD Kuru

Animal prion disease Sporadic Scrapie in sheep CWD in mule deer and elk Infectious

Genetically linked mutations to the Prnp gene

vCJD: infection possibly from contaminated cow products iCJD: infection from prion-contaminated HGH, dura mater grafts, and surgical tools Kuru: infection from ritualistic cannibalism among the Fore people of Papua New Guinea

Spontaneous conversion of PrPc to PrPsc?

Possible horizontal and vertical transmission; contaminated MBM feed; and unknown reasons

Scrapie in sheep BSE CWD in the wild Exotic ungulate spongiform Encephalopathy Scrapie in experimental mice Transmissible mink encephalopathy Feline spongiform encephalopathy sCJD, sporadic Creutzfeldt-Jakob disease; iCJD, iatrogenic Creutzfeldt-Jakob disease; fCJD, familial Creutzfeldt-Jakob disease; vCJD, variant CreutzfeldtJakob disease; GSS, Gerstmann-Straussler-Scheinker syndrome; sFI, sporadic fatal insomnia; fFI, familial fatal insomnia; BSE, bovine spongiform encephalopathy; CWD, chronic wasting disease; HGH, human growth hormone.

months to decades, and pre-mortem biopsy is not available for diagnosis (Serban et al., 1990; Hill et al., 1999). With recent evidence pointing to possible trans-species infection via ingestion, possible contamination of human food supplies has increased concerns over food safety. This review summarizes recent findings regarding the role of metals in physiological functions of PrPc proteins as well as the pathophysiology of prion diseases. A special emphasis has been focused on the role of the transition metals such as copper and manganese in the disease process. 1.1. Prion diseases PrPc sequence is very well conserved throughout the mammalian species, which may explain its capacity for possible interspecies transmission (Hill et al., 2000; Horiuchi et al., 2000).

The close homology (>90%) between different species is presented in Fig. 1. Nevertheless, some significant differences do exist between species in certain regions of PrPc, such as the number of octapeptide repeats. Although each species has a distinct form of prion disease, infection can be transmitted from one species to another (Priola and Lawson, 2001). Studies with deletion mutants have shown that the whole protein may not be necessary for the infection and propagation of the disease (Supattapone et al., 1999). It is believed that there is a region that has a direct physical interaction between the PrPc and PrPsc, allowing for the PrPsc to act as a template for the conformational change of PrPc into PrPsc, and this region is believed to be crucial in the formation of PrPsc from PrPc (Telling et al., 1996). The heritable, transmittable and sporadic nature of prion disease is quite interesting. About 20 mutations have been reported in the PrP gene that is linked to the inherited human prion disease (Collinge et al., 1989; Goldgaber et al., 1989; Hsiao et al., 1989). Each of the human forms of the prion disease has very distinct pathologies and symptoms. The sporadic, familial, iatrogenic, and new variant forms of CJD are characterized by spongiform degeneration of the gray matter and accumulation of protease resistance PrPsc in the gray matter, with little or no PrP amyloid plaque formation, with the exception of the new variant CJD (Prusiner and DeArmond, 1990; Haltia et al., 1991; Liberski et al., 1991; Budka et al., 1995). And CJDs are characterized by dementia and ataxia (Budka et al., 1995). Kuru, the prion disease of the Fore people who practiced ritualistic cannibalism in the New Guinea region, is highly similar in pathology and symptoms to CJD. Kuru is characterized by damage to the central nervous system similar to that of CJD, but kuru is also associated with severe atrophy of the cerebellum (Prusiner, 1989; Brown and Gajdusek, 1991; Prusiner and DeArmond, 1991; Liberski and Brown, 2004). Both familial fatal insomnia (fFI) and sporadic fatal insomnia (sFI) are characterized by pathological damage similar to that of CJD, as well as progressive insomnia (Reder et al., 1995; Tateishi et al., 1995; Chapman et al., 1996; Silburn et al., 1996). GSS is pathologically different from the other human prion disease in its multi-centric PrP positive amyloid plaques, highly truncated forms of PrP, some protease sensitive PrPsc, and later onset of dementia (Hsiao and Prusiner, 1991; Giaccone et al., 1992; Hsiao et al., 1992; Barcikowska et al., 1993). Whether inherited, transmitted, or sporadic in nature, the PrPsc from the brain of diseased individuals are infectious, and can be transmitted horizontally (Hogan et al., 1999; Zobeley et al., 1999; Flechsig et al., 2001; Ricketts and Brown, 2003). Of the greatest concern, prion disease has reportedly been transmitted from blood transfusions (Brown, 2005a,b). The prion diseases affecting animals, i.e. scrapie, bovine spongiform encephalopathy (BSE), chronic wasting disease (CWD), and transmissible mink encephalopathy (TME), share some of the common characteristic pathologies that have been described for CJD in humans (Westaway et al., 1995; Collinge, 2001; Hill and Collinge, 2003). Naturally occurring cases of scrapie have been identified in Europe for the last 200 years (Brotherston et al., 1968), and more recently, naturally occurring cases of CWD have been found in elk and mule deer in North America (Williams and Young, 1992; Guiroy et al., 1993).

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Fig. 1. PrPc homology among mammalian species. PrPc homology was obtained from the NCBI Entrez protein database. Yellow corresponds to highly conserved regions in the species listed, while blue corresponds to moderately conserved regions, and white corresponds to less conserved regions.

Although the exact mechanism of infection is not clear, experimental infections can occur between various species. A species barrier does exist in disease transmission among various species; i.e. CWD in mule deer to cattle, scrapie to elk (Hamir et al., 2004, 2005); such species barriers prolong the incubation period required for the disease. However, after subsequent passages within the same species, the species barrier is overcome and the incubation period of the disease is no longer prolonged (Prusiner, 1993; Priola, 1999; Hill et al., 2000). 1.2. Structure of PrPc PrPc is a highly conserved protein among the mammalian species (Martins and Brentani, 2002). It is a surface protein,

predominantly found in the CNS and several other cell types, linked via a glycosyl phosphoinositol (GPI) anchor, with two N-linked glycosylations (McKinley and Prusiner, 1986). PrP gene is found on chromosome 20 in humans and encodes for 254 amino acid sequences before processing (Fig. 2). The first 22 amino acid residues of the N-terminus are cleaved shortly after translation, and the last 24 amino acid residues of the C-terminus are cleaved prior to the addition of the GPI anchor. PrPc contains several octapeptide repeat sequences (PHGGSWGQ) toward the N-terminus which have binding affinity for divalent metals such as copper, zinc, nickel and manganese, with preferential binding for copper (Hornshaw et al., 1995; Viles et al., 1999). Although it is not clear as to whether any divalent cation could bind to this site, it is strongly

Fig. 2. Schematic of PrPc. PrPc molecule is 254 amino acids in length, with N-terminal and C-terminal sequences that are cleaved shortly after translation. GPI anchor is attached to the C-terminus post cleavage of the signal sequence, and addition of two N-linked glycosylations occurs at Asn 181 and 197. Three a-helices are present at 144–157 (a1), 172–193 (a2), and 200–227 (a3). A disulfide bridge is formed between a2 and a3 at Cys 179 and 214, respectively. Two small b-sheets are also present at 129–131 (b1) and 161–163 (b2). Toward the N-terminus is the octapeptide repeat region (PHGGSWGQ) which has His residues suggested to play a role in metal binding. Additional metal binding sites His 96 and 111 are also present.

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speculated that this site is crucial for prion activity and important in the etiology of the prion disease. The number of octapeptide repeats differs from species to species, and a higher number (>24) of repeats has been associated with the human form of the prion disease known as Gerstmann-Stra¨usslerScheinker (GSS) syndrome (Palmer et al., 1993). More recently, higher affinity metal binding sites have been identified at His 96 and 111 (Jackson et al., 2001; Jones et al., 2004), but the exact role of these higher affinity metal binding sites remains elusive. Other studies have suggested that most of the N-terminus is rather unstructured, while the C-terminus is highly structured, with two a-helices linked via disulfide bond, a shorter a-helix, and two short strands of b-sheet structures (Viles et al., 2001; Renner et al., 2004; Di Natale et al., 2005; Jones et al., 2005a). Binding of the divalent cation to the octapeptide repeats has been speculated to facilitate the folding of the largely unstructured N-terminus, thereby stabilizing the protein conformation (Cereghetti et al., 2003). Properly folded prion protein is present on lipid membrane rafts, and is believed to be internalized via clathrin-mediated endocytosis with a half-life of about 6 h (Peters et al., 2003; Prado et al., 2004). Internalized PrPc and abnormally folded protein is rapidly degraded via the ubiquitin proteasomal system (Laszlo et al., 1992; Ciechanover and Brundin, 2003; Kang et al., 2004). 1.3. Conformational changes in PrPc Changes in conformation into the infectious prion protein (PrPsc) from PrPc result from dramatic change in the secondary structure, with increased b-sheet content, and decreased ahelices content (Gasset et al., 1993; Huang et al., 1996a). These altered conformations tend to aggregate into insoluble fibrils and are resistant to proteinase-K digestion (Turk et al., 1988; Martins and Brentani, 2002; Yakovleva et al., 2004). Not only are the PrPsc proteins proteinase-K resistant, these abnormal forms of protein are not inactivated by normal procedures that modify nucleic acids (Prusiner et al., 1980; Diener et al., 1982; Prusiner, 1982). There are two different models for the prion conversion from PrPc to PrPsc. One is the heterodimer model, in which a single particle of PrPc interacts with PrPsc to form more PrPsc (Prusiner et al., 1988). The other model is the nucleated polymerization model, in which the oligomer or short polymers of PrPsc, instead of a single particle of PrPsc, interact with PrPc to form more PrPsc (Cohen et al., 1994; Huang et al., 1996b). There has been some speculation about an additional chaperone protein requirement of protein X for propagation, while others have maintained that imbalance of metal binding may be the key factor in the formation and propagation (DeArmond and Prusiner, 1995; Telling et al., 1995). However, the synthetic form of the prion protein, even in its abnormal conformational folding, does not induce prion disease in animals (Nguyen et al., 1995; Kaneko et al., 1997). Recently, Prusiner’s group induced neurological disease in transgenic mice, and not in wild-type mice, with synthetic prion proteins (Legname et al., 2005). Thus, other proteins found endogenously in the cell may be needed for folding of the PrPsc.

1.4. Physiological functions of PrPc Although PrPc is highly expressed in CNS, the biological role is not well established. The prion protein has been speculated to function as an antioxidant, a metal transporter, a cell adhesion molecule, and a signal transducer (Martins et al., 2001; Martins and Brentani, 2002; Wechselberger et al., 2002; Lasmezas, 2003; Deignan et al., 2004). A number of studies performed with prion-knockout cells and mice have shown that the PrPc lacking phenotypes were more sensitive to oxidative stress (Brown et al., 1997a; Brown et al., 2002; Sakudo et al., 2004). Perturbations to various intracellular antioxidant systems such as glutathione levels, glutathione reductase activity, glutathione peroxidase activity, and superoxide dismutase (SOD) activity have been observed (Brown et al., 1997a). Furthermore, infection with PrPsc has also been implicated to increase the susceptibility to oxidative stress (Wong et al., 2001). Others have speculated that the prion protein functions as a metal transporter (Whittal et al., 2000; Opazo et al., 2003). Comparison of various metal levels in the brain of prion protein knock-out versus wild-type mice has shown significant differences in the amount of metals present in the brain (Wong et al., 2001; Thackray et al., 2002; Brown, 2003). Similarly, those mice infected with scrapie have altered brain metal content as well (Wong et al., 2001; Thackray et al., 2002; Fernaeus et al., 2005b). Differences in iron, copper, as well as manganese levels were observed as compared to controls in these studies. A number of study results have implicated the associative role of the PrPc with various cellular adhesion molecules, which are important in the role of cell proliferation and survival. PrPc has been reported to interact with such surface cell adhesion molecules as neural cell adhesion molecule (NCAM) and laminin (Zanata et al., 2002; Santuccione et al., 2005). Additionally, since PrPc is localized to the lipid rafts, a role in signal transduction has been hypothesized (Mouillet-Richard et al., 2000; Reilly, 2000). Recent studies have shown that PrPc has a role in neurite outgrowth and survival of neurons as well (Chen et al., 2003; Santuccione et al., 2005). Interestingly, cytoplasmic polyadenylation element binding protein (CPEB), a protein involved in synaptic plasticity and long-term memory, has been found to self-replicate in a prion protein like manner (Darnell, 2003; Si et al., 2003). There has been an increased interest in recent years in the investigation of the role of prion proteins in memory function (Bailey et al., 2004; Papassotiropoulos et al., 2005; Shorter and Lindquist, 2005). Furthermore, cytosolic accumulation of PrPc has been shown not to be toxic but rather to protect human neuronal cells against Baxinduced cell death (Roucou et al., 2003). Another group has shown that cytosolic accumulation of PrPc to be highly toxic, and distinctly different than PrPsc (Ma et al., 2002). Alternatively, PrPc has been proposed to function in various neuroprotective signaling pathways (Chiarini et al., 2002). A recent study has shown that cytosolic PrPc is not toxic to N2a mouse neuroblastoma cells and primary neurons expressing the pathogenic form of PrP mutants (Fioriti et al., 2005). We have recently shown that PrPc can protect against oxidative damage

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in cell culture models (Anantharam et al., 2004). Although a clear consensus has not been reached with regard to the exact function of the prion proteins, understanding the normal function will help elucidate the pathogenesis of prion disease. 1.5. Role of copper in the physiological function and pathogenesis of the prion protein Copper is an essential transitional metal required for activity of multiple mammalian enzymes (Wysokinska, 1964; O’Dell, 1976). Through its ability to exist in various redox states, copper is able to function as a catalyst for enzymatic function. However, since it can exist in multiple redox states, perturbance to the homeostasis of copper results in a number of detrimental effects (Aust et al., 1985). Free copper rarely exists in organisms, which may be due to its highly regulated uptake, sequestration, and excretion. When this highly regulated state is disturbed, copper associated CNS disorders are prevalent, such as Wilson’s disease, Menke’s disease, Alzheimer’s disease, and the more recently identified prion disease (Camakaris et al., 1999). 1.6. Copper and PrPc It is well established that PrPc binds with copper, and PrPc can be isolated with copper affinity columns (Muller et al., 2005; Todorova-Balvay et al., 2005). Numerous studies have used recombinant prion protein to measure the binding affinity of different amino acid sequences of prion protein to copper (Table 2). Although the proposed binding affinities differ, most researchers would agree that copper binds specifically to the octapeptide region toward the N-terminus of the prion protein (Hornshaw et al., 1995; Aronoff-Spencer et al., 2000). In general, binding of metals to the octapeptide region reduces copper ion in a pH dependent manner (Miura et al., 2005). As aforementioned, additional copper binding sites with higher binding affinities have been identified above the octapeptide region (Jones et al., 2004). The octapeptide region is crucial to metal ion-induced endocytosis of the prion protein (Perera and Hooper, 2001; Brown and Harris, 2003). Also, the high conservation of the metal binding regions among various mammalian species indicates a correlative role between copper and the prion protein. Interestingly, brain from prion protein knock-out mice appears to contain significantly less copper as compared to brain from wild-type mice (Brown et al., 1997a; Brown et al., 1998). Likewise, PrPsc-infected mice have significantly lower levels of copper compared to brains of wildtype mice (Wong et al., 2001; Thackray et al., 2002). This Table 2 Copper binding affinity studies Kd for copper (II)

Sequences examined

Authors

6.7 mM 5.9 mM 14 mM 10 14 M and 4  10

60–91 23–98 23–231 52–98 and 91–231

Hornshaw et al. (1995) Brown et al. (1997a,b) Stockel et al. (1998) Jackson et al. (2001)

14

M

781

evidence suggests a role of the prion protein as a possible copper transporter in the CNS. Additional studies have shown that over-expression of PrPc resulted in higher binding of copper in the membrane fraction, which further supports the possible notion that PrPc is a copper transporter (Herms et al., 1999; Stuermer and Plattner, 2005). PrPc may be concentrated in the synapse of neurons, suggesting that prion protein may regulate copper levels at the synaptic junction, possibly by acting as a metal sink (Nishimura et al., 2004). However, disturbance of the copper regulating function of PrPc at the neuronal synapse could result in neuronal susceptibility to oxidative damage as a result of excessive free copper. A more recent study indicated that PrPc may not be responsible for copper uptake in the synaptosomes (Giese et al., 2005). Together, these findings strongly suggest that copper–PrPc interactions may be more complex than originally perceived. 1.7. Effect of copper on PrPc expression Additional evidence supports the role of copper in the physiological control of PrPc expression. Recent results showed that copper could modulate PrPc expression by reducing PrPc mRNA, as well as total PrPc, found in cell lysates (Toni et al., 2005). Along with these changes in PrPc expression, the amount of PrPc shed into the extracellular media was greatly increased. This effect on PrPc by copper was abolished by co-treatment with the copper chelator cuprizone, indicating that these events required extracellular copper. Another study has shown that treatment with copper dramatically increased PrPc levels in neuronal cells (Varela-Nallar et al., 2005). Likewise, cotreatment with the copper chelating compound bathocuprionedisulfate (BCS) returned PrPc levels to normal. The increased prion protein expression was shown to be specific for copper and cadmium, and not for any other metals. The capability of copper to modulate the expression levels of PrPc would indicate a functional role of copper in the physiological function of PrPc. 1.8. Role of copper in physiological function of PrPc as an antioxidant The role of the prion protein in the brain is not as well defined as other synaptic proteins. Not only does the PrPc seem to act as a copper transporter, but studies have also shown PrPc has superoxide dismutase (SOD)-like activity, enabling its effective functioning as an antioxidant in CNS (Brown et al., 1997b). Studies with recombinant prion protein showed proper protein folding in the presence of copper, endowing PrPc with antioxidant capacities. Further, two atoms of copper bound to PrPc were sufficient for SOD-like activity (Stockel et al., 1998). However, this SOD-like function of PrPc was demonstrated in in vitro simulations, and studies have refuted the possibility that prion protein functions as an SOD-like protein in vivo (Hutter et al., 2003). Studies with PrP knock-out mice have shown decreased SOD activity, not to mention increased susceptibility to oxidative stress (Brown et al., 1997a; Brown and Besinger, 1998). These observations strongly indicate PrPc functions increase antioxidant levels indirectly or directly. One theoretical view is that the

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low binding affinity of PrPc to copper would preclude its ability to function as metal-associated enzymes (Jones et al., 2005b). Other groups have shown that the prion protein does not have SOD-like activity in vivo or in vitro (Hutter et al., 2003; Jones et al., 2005b). Although the role of the PrPc as a direct acting SOD or as an indirect oxidative stress reducer is not yet definitive, PrPc clearly has some protective role in CNS. 1.9. Role of copper in pathogenesis of prion disease Copper has been shown to have the capacity to convert PrP into protease resistant and detergent insoluble forms in certain in vitro conditions (Quaglio et al., 2001) and to inhibit formation of fibrils from recombinant PrP (Bocharova et al., 2005). Although some of these biochemical changes are commonly associated with initial stages of PrPsc formation, additional studies have shown that copper-induced biochemical changes were distinct and reversible. Interestingly, copper can also modulate the protease resistance of PrPsc molecules (Nishina et al., 2004). A study showed that PrPsc formed in the absence of copper was 20 times more susceptible to proteinaseK digestion, whereas addition of copper reinstated the normal protease resistance. Additionally, a study with protein misfolding cyclic amplification (PMCA) showed that PrPsc with copper could propagate and form more PrPsc from PrPc in the presence of deoxycholic acid (Kim et al., 2005). Additional support for the role of copper in the pathogenesis of prion disease is that chelating copper delays the onset of the disease (Sigurdsson et al., 2003). Earlier studies have shown that chelating copper (using cuprizone) resulted in spongiform-like degeneration in the animal brain (Blakemore, 1972; Kimberlin and Millson, 1976). Of note, homeostatic balance of copper in the CNS is crucial for normal function, and these studies clearly support the role of copper with PrPc in the pathogenesis of prion disease. However, a number of questions still need to be answered: is copper needed in the normal functioning of the PrPc? What are the cellular mechanisms underlying copperinduced conversion of PrPc to PrPsc? Furthermore, is copper required for increased susceptibility to infection? 1.10. Manganese and PrPc Manganese is also an essential trace element crucial for survival that is found abundantly in nature. However, manganese imbalance can lead to detrimental disorders of the CNS, for review see Dobson et al. (2004). Studies have shown that manganese, like copper, can bind to PrPc and can also be used for formation of PrPsc from PrPc via the PMCA technique (Kim et al., 2005). Interestingly, mice infected with PrPsc have dramatic differences in blood, muscle, and brain metals content as compared to the non-infected mice (Thackray et al., 2002). These infected mice showed increased manganese and decreased copper content, suggesting that during the course of disease infection, altered metal content could be the initial signs of infection, or the altered metal content could be due to the infection itself. Recombinant PrPc has been shown to bind to PrPc in vitro, and when refolded in the presence of manganese,

proteinase-K resistant forms of PrPc were found (Brown et al., 2000). These forms were not pathogenic, yet interestingly, PrPc binding with a different metal could potentially alter the secondary structure of a protein. These PrPc refolded in the presence of manganese retained some SOD-like activity similar to copper bound PrPc, but to a lesser capacity. We found that PrPc knock-out mouse neural cells retained more manganese after manganese treatment, and were more susceptible to manganeseinduced toxicity compared to prion expressing mouse neural cells (unpublished observations). 1.11. Iron and PrPc Iron is probably one of the most important metals required for survival. It is highly regulated in physiological systems to maintain balance. Free iron does not exist in physiological systems, and is always found bound to various iron transporting proteins and iron bound enzymes. More recently, Fernaeus et al. demonstrated that iron regulation is disturbed in scrapie infected mouse neuroblastoma cells (Fernaeus and Land, 2005; Fernaeus et al., 2005a,b). Apparently, normal processing of iron by these cells was interrupted by infection, resulting in decreased intracellular iron levels. The presence of additional iron resulted in increased reactive oxygen species formation and cell death. These findings suggest that scrapie infection alters iron metabolism, and decreases the cell’s capacity to deal with excessive iron. Also, these findings suggest that cells have a very small tolerance to iron disturbance, since the scrapie infection consists of only a small percentage of cells. A recent study demonstrates that the iron binding protein ferritin forms a complex with a fragment PrPsc to enhance the transport of PrPsc in an intestinal endothelial cell model (Mishra et al., 2004), suggesting a role for ferritin in transport of infected prion protein across the intestine. 1.12. Role of other metals in the physiological function and pathogenesis of the prion protein Other divalent cations such as manganese, iron, zinc, nickel, and many others may have the potential to interact with PrP. Table 3 shows the effect of different metals on PrPc function. While binding studies have shown that only manganese, zinc, and nickel have higher binding affinities, other metals could have a role in physiological function, or in alteration of the normal function of PrP (Gaggelli et al., 2005). Nickel (II) has been used in metal affinity columns to isolate PrP (Yin et al., 2003). Metal imbalance in the brain has been hypothesized to cause prion disease (Wong et al., 2000; Thackray et al., 2002). Metal imbalance could occur through aging, environmental sources or even from nutritional deficiencies. To investigate the influence of environmental factors on sporadic forms of prion disease, evaluation of the correlation between increased prion disease and soil mineral levels has been proposed (Purdey, 2000; Chihota et al., 2004; Purdey, 2004). PrPc may be specific for copper, but similarities in ionic strength, hydration size, and valence may allow other metals to bind to PrPc if present in sufficient concentrations.

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Table 3 Metals and their role in prion protein function Metals

Result

References

Copper

Superoxide dismutase like activity

Conversion into PK resistant forms distinct from scrapie Delays onset of disease Inhibits conversion into amyloid fibrils Regulate expression and metabolism

Brown and Besinger (1998), Brown et al. (2000), Wong et al. (2000), Brown et al. (2001), and Rachidi et al. (2003) Pauly and Harris (1998), Brown and Harris (2003), and Haigh et al. (2005) Quaglio et al. (2001) and Kuczius et al. (2004) Sigurdsson et al. (2003) and Hijazi et al. (2003) Bocharova et al. (2005) Toni et al. (2005) and Varela-Nallar et al. (2005)

Iron

Formation of PrPres with PMCA

Kim et al. (2005)

Manganese

Amplify aggregation of PrP; pro-aggregatory effect Formation of PrPres with PMCA Increase resistance to proteinase K digestion

Giese et al. (2005) and Levin et al. (2005) Kim et al. (2005) Brown et al. (2000)

Zinc

Induce rapid turnover of PrP Critical for PrP 106–126 aggregation and neurotoxicity

Brown and Harris (2003) Jobling et al. (2001)

Stimulate endocytosis; increase protein turnover

2. Conclusion The etiology of prion disease and the normal physiological functions of the PrPc remain to be answered. Mounting evidences point to the role of copper in the antioxidant role of the PrPc and metal imbalance in prion disease. Although most of these trace element metals are needed in maintenance of normal physiological conditions, perturbations of these metal levels in the brain could have detrimental effects. A hypothetical model depicting the role of metals in the conversion of PrPc to PrPsc is shown in Fig. 3. Inside the cell, PrPc and PrPsc could exist in equilibrium, but the level of PrPsc could be below the detectable limits. Environmental exposure to divalent cations including manganese may induce

structural and/or functional changes thus affecting the equilibrium. Additionally, these metals could potentially inhibit proteasomal activity and thereby increase the PrPsc levels. Alternatively, metals could displace the copper from the PrPc protein and potentially alter the structure to an intermediate form which is partially rich in b-sheet. The b-sheet rich intermediate PrPc form could eventually be converted to PrPsc. The metals could also bind to monomeric PrPsc to propagate the aggregation of the toxic prion protein. Therefore, it is imperative to investigate the interactions of metals with cellular prion proteins in order to better understand not only the normal function of the PrPc in the nervous system but also the pathogenic mechanisms associated with conversion of PrPc to PrPsc. A detailed understanding of the role of metals in prion

Fig. 3. Schematic model of metal-induced changes in the structure and function of PrPc. Normal prion protein (PrPc) and an abnormal form (PrPsc) could exist normally in equilibrium, with the possibility that PrPsc is normally degraded to undetectable levels. Various metals such as manganese, cadmium and zinc could potentially inhibit proteasomal activity, thereby increasing the amount of PrPsc levels to become detectable. Alternatively, displacement of copper binding to PrPc with manganese could potentially alter the structure into an intermediate form with a higher content of b-sheets. With replacement of copper, these would revert back to PrPc forms. There also would have to be another event where partially b-sheet rich PrPc could turn into PrPsc forms. In turn, the formation of PrPsc would aggregate together in the presence of excessive manganese, thereby becoming toxic to neuronal cells.

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