Is there a role for copper in neurodegenerative diseases?

Molecular Aspects of Medicine 26 (2005) 405–420 www.elsevier.com/locate/mam

Review

Is there a role for copper in neurodegenerative diseases? Waldo Cerpa a, Lorena Varela-Nallar a, Ariel E. Reyes b, Alicia N. Minniti a, Nibaldo C. Inestrosa a,* a Centro de Regulacion Celular y Patologı´a ‘‘Joaquin V. Luco’’ (CRCP), MIFAB, Facultad de Ciencias Biologicas, Pontificia Universidad Catolica de Chile, Alameda 340, Santiago, Chile b Millennium Nucleus in Developmental Biology (MNDB), Facultad de Ciencias de la Salud, Universidad Diego Portales, Av. Eje´rcito Libertador 141, Santiago, Chile

Abstract Copper is an essential metal in living organisms; thus, the maintenance of adequate copper levels is of vital importance and is highly regulated. Dysfunction of copper metabolism leading to its excess or deficiency results in severe ailments. Two examples of illnesses related to alterations in copper metabolism are Menkes and Wilson diseases. Several proteins are involved in the maintenance of copper homeostasis, including copper transporters and metal chaperones. In the last several years, the b-amyloid-precursor protein (b-APP) and the prion protein (PrPC), which are related to the neurodegenerative disorders Alzheimer and prion diseases respectively, have been associated with copper metabolism. Both proteins bind copper through copper-binding domains that also have been shown to reduce copper in vitro. Moreover, this ability to reduce copper is associated with a neuroprotective effect exerted by the copper-binding domain of both proteins against copper in vivo. In addition to a functional link between copper and b-APP or PrPC, evidence suggests that copper has a role in Alzheimer and prion diseases. Here, we review the evidence that supports both, the role of b-APP and PrPC, in copper metabolism and the putative role of copper in neurodegenerative diseases.
2005 Elsevier Ltd. All rights reserved.

*

Corresponding author. Tel.: +56 2 6862720; fax: +56 2 6862959. E-mail address: [email protected] (N.C. Inestrosa).

0098-2997/$ - see front matter
2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.mam.2005.07.011

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Abbreviations: APP, Amyloid-precursor protein; PrPC, Cellular prion protein; COX, Cytochrome c oxidase; SOD, Superoxide dismutase; ROS, Reactive oxygen species; AD, Alzheimer disease; ALS, Amyotrophic lateral sclerosis; CJD, Creutzfeld–Jakob disease; CSF, Cerebrospinal fluid; Ctr1, Copper transporter protein 1; CuBD, Cu binding domains Keywords: Copper; Copper homeostasis; Ctr1; APP; Prion protein

Contents 1. 2.

3.

4.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Copper transport and metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Proteins involved in copper homeostasis . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. New players in copper metabolism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Copper and neurodegenerative diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Role of copper in Alzheimer disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Role of copper in prion diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Metals such as copper (Cu), zinc, iron and molybdenum are essential for living organisms. Copper is necessary for the activity of a number of physiologically important enzymes, the majority of which catalyze redox reactions (Miranda et al., 2000). As a cofactor in multiple redox reactions, Cu is also involved in the production of potentially damaging radical species through Fenton or Haber–Weiss reactions (Miranda et al., 2000). For this reason, all organisms have redundant mechanisms for controlling Cu concentrations. Among those recently elucidated, sequestration of the metal and its delivery to specific cellular compartments appear important. Either a deficiency or an excess of Cu can result in serious consequences to the organism (Fig. 1). When the Cu concentration is insufficient, cells do not have enough ‘‘metallic raw material’’ for enzyme production since transition metals such as Cu and zinc are important for a functional catalytic center. The decreased production of active enzymes leads to a decline of metabolic activity. For example, cytochrome c oxidase (COX), involved in energy metabolism, is negatively affected by unusually low Cu and zinc concentrations since its catalytic center is inactive under these conditions. Therefore, the cell is unable to carry out this essential metabolic activity (DiMauro et al., 1990). The enzymes involved in energy metabolism are not the only such molecules affected by metal imbalance. The activities of those responsible for the removal of cellular free radicals are greatly affected when the available Cu is decreased. The clearest case for this is that of superoxide dismutase

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Fig. 1. Effects of copper deficiency or excess. Region 1 shows that low concentrations of copper cause adverse effects, as copper is essential for enzyme synthesis. Region 2 represents intermediate copper concentrations that are adequate for enzyme synthesis and function. These concentrations are organismdependant. Region 3 shows that negative effects appear when copper concentrations are high (discussed in the text).

(SOD), which has Cu and zinc at its catalytic center and requires Cu for its synthesis (Rosen et al., 1993). On the other hand, an excess of Cu is associated with oxidative stress and can be toxic at both the cellular and organismic level. Cu is generally found in its bivalent state (Cu2+), but, when in its monovalent form (Cu1+), it is able to transfer one electron and generate reactive oxygen species (ROS) such as hydroxyl radicals (Halliwell and Gutteridge, 1984). These radicals are responsible for cellular damage that includes protein oxidation, lipid peroxidation in membranes and DNA damage. In recent years, research on the role of nitric oxide (NO) in neurons has led to the identification of NO stress derivatives. The reaction between superoxide and NO generates peroxynitrite, a strong oxidant species able to produce highly reactive intermediates by the Fenton reaction plus hydrogen peroxide (Wang et al., 2003). ROS generation is related to aging, cancer and neurodegenerative disorders (Harrison et al., 2000). In patients with Alzheimer disease (AD), amyotrophic lateral sclerosis (ALS) and Creutzfeld–Jakob disease (CJD), the levels of ROS are increased in specific brain regions, especially in the lesions of affected areas (Miranda et al., 2000). ROS generation is a normal cellular process but its imbalance is directly associated with the appearance of diverse pathologies. In this context, metals become crucial in the direct modulation of ROS generation and participation in pathological processes. It is important to emphasize that metals not only modulate the appearance of ROS, but also participate in other processes that indirectly affect ROS appearance. Two well-characterized human genetic disorders that serve as examples of the consequences of Cu imbalance are Menkes and Wilson disease (Bull et al., 1993). In Menkes disease, the transport of dietary Cu from intestinal cells is impaired, leading to low serum Cu levels, while in Wilson disease there is a defect in cellular Cu export which leads to the accumulation of high levels of copper. The genes for Menkes and Wilson disease share 55% amino acid homology, but their tissue-specific

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expression differs. Menkes disease affects 1/50,000 to 1/250,000 live births and is a serious X-linked disorder caused by the dysfunction of the ATPase ATP7A. This protein is an ATP-dependent Cu transporter that is found across species from bacteria (Odermatt et al., 1993) to humans (Sambongi et al., 1997). It is localized in the trans Golgi network (TGN) but is relocated to the plasma membrane when the cell is exposed to Cu (Strausak et al., 1999). Due to its essential function, ATP7A is found in many cell types including neurons and glia, as well as in several tumor cell lines including the neuronal cell lines PC12 and C6. That those affected by Menkes disease suffer neuronal disorders is not unexpected as the enzymatic activities related to chemical detoxification are greatly reduced (Hartmann and Evenson, 1992). The enzymatic activities that are most affected are those corresponding to Cu dependent-SOD and COX, two enzymes that play significant roles in controlling the production of ROS during active neuronal metabolism (Menkes, 1988). A decrease in activities of these two enzymes likely accounts for the majority of the neurological symptoms. Wilson disease affects 1/100,000 live births and is an autosomal recessive disorder resulting in Cu poisoning. It affects the liver, kidneys, eyes and brain and can lead to death. This occurs as a result of mutations in the ATP7B Cu transporter gene involved in Cu excretion (Dijkstra et al., 1996). ATP7B participates in the transport of apo-ceruloplasmin from the TGN and late endosomes to the plasma membrane (Nagano et al., 1998). Contrary to what occurs in Menkes disease, in Wilson disease the blood concentration of Cu becomes unusually high when the liver, damaged by Cu accumulation, releases the metal directly into the bloodstream. Thus, Cu ions are carried throughout the body, damaging other organs. Wilson patients with neurological symptoms show elevated Cu levels in cerebrospinal fluid (CSF) (Kodama et al., 1988).

2. Copper transport and metabolism 2.1. Proteins involved in copper homeostasis Cu uptake by the gut/intestine and its transport to the entire organism requires the function of several proteins, including: metal transporters, chaperones, and metallothioneins. After the metal has been taken up by the enterocytes it is exported to the bloodstream, a step that involves cytoplasmic transport to the membrane and exocytosis. Cu is then distributed to the rest of the tissues and is taken up by the cells. Once inside the cell, Cu homeostasis needs to be tightly regulated; the metal is complexed with other proteins to avoid the formation of deleterious hydroxyl radicals. Copper uptake from the extracellular medium depends on the specific Cu transporter protein 1 (Ctr1), originally discovered in yeast as a high affinity Cu transporter (Dancis et al., 1994). Recent studies in transgenic mice have demonstrated that Ctr1 is essential for the survival of mammalian embryos (Kuo et al., 2001). Ctr1 is located in the plasma membrane of cells and appears to be ubiquitously expressed in mam-

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malian tissues (Klomp et al., 2002). Ctr1 takes Cu2+ from the extracellular space and delivers Cu1+ within the cell, probably by the action of a Cu reductase present at the cell surface (Opazo et al., 2003; Suazo et al., in press). Under conditions of low extracellular Cu concentration, Ctr1 turnover appears stable, but high Cu concentrations trigger internalization and degradation of Ctr1 without affecting other membrane proteins (Dancis et al., 1994). Besides Ctr1, other transporters that transfer Cu into the cell have been described in mammals, including: a divalent metal transporter 1 (DMT1), also known as natural resistance associated macrophage protein 2 (Nramp 2): and a divalent cation transporter 1 (DCT1) originally identified with iron uptake. Recently, Ctr1 was cloned in zebrafish (Mackenzie et al., 2004); the translated protein showed 70% homology with the human form of hCtr1. Functional studies show that Ctr1 is also essential for development in zebrafish. Interestingly, Ctr1 appears ubiquitously expressed during early stages of zebrafish development (Fig. 2A). However, this expression becomes restricted to tissues such as brain, notochord (peripheral nervous system) (Fig. 2B and C), liver and gut in later development. These observations suggest a fine regulation of Ctr1 expression during development and organogenesis. The 5 0 flanking region of zebrafish Ctr1 contains five metal responsive elements (MRE) and eight binding sites for heat shock factor 1 (HSF1) (AER, unpublished data). At present, the regulation of Ctr1 expression is uncertain even though expression of this protein appears to be important in the uptake of Cu (Harris, 2003) and in sensitivity to platinum drugs (Safaei and Howell, 2005). Cuprous (Cu1+) ions are rapidly routed to the three chaperone pathways: Cu dependent enzymes in the cytosol, mitochondria and the secretory pathway, to avoid the formation of ROS. Then metallochaperones deliver Cu to the target cuproenzymes. Several cytoplasmic chaperones such as Atox1, Cox17 and Cu chaperone for SOD (CCS) have been shown to deliver Cu to specific cuproenzymes. Atox1 (human Atx1 homologue, HAH1), a mammalian homolog of yeast Atx1 (Klomp et al., 1997), specifically transports Cu to the P-type ATPases, ATP7A (Hamza et al., 1999) and ATP7B (Hamza et al., 1999), by the secretory pathway. These Cu-ATPases concentrate in the TGN where they join enzymes such as tyrosinase and ceruloplasmin. Genes from Caenorhabditis elegans and Arabidopsis also encode for proteins very similar to Atx1. In addition, Cox17p is necessary to deliver Cu to COX in the mitochondria, a step that is critical for the assembly of the yeast COX enzyme complex. Comparative studies of Cox17p homolog from humans, mice and pigs suggest similar functions. CCS is known to transfer the Cu that activates cytoplasmic SOD 1, and does not deliver Cu to proteins in the mitochondria, nucleus or secretory pathway (Harris, 2003). Recently, the subcellular localization of Ctr2, a member of the Ctr1 family (with yeast Ctr1 and Ctr3 as high affinity Cu transporters at the plasma membrane) has been characterized. This transporter has been suggested to be a low affinity Cu transporter. However, studies in yeast have established that Ctr2 is located in the vacuolar membrane and its function is to transport Cu from the vacuole to the cytoplasm, providing Cu to all three of the Cu chaperone pathway and identifying a novel mechanism for Cu mobilization (Rees et al., 2004).

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Fig. 2. Localization of the copper transporter, Ctr1, mRNA in zebrafish embryo. Distribution of the Ctr1 mRNA analyzed at different stages of embryo development using whole mount in situ hybridization. (A) 16 h post-fertilization (hpf), (B) 24 hpf, (C) 40 hpf and (D) 3.5 days post-fertilization. Ctr1 is expressed at all stages examined as shown by in situ hybridization. In early embryogenesis, expression is homogeneously distributed (A), later the mRNA becomes localized to the central and peripheral nervous system (B to D) and ventral body and is found predominantly in the intestinal region (D). Zebrafish embryos lateral view, anterior at right. (Adapted from Mackenzie et al., 2004).

2.2. New players in copper metabolism The main proteins associated with Alzheimer and prion diseases (amyloid-precursor protein (APP) and prion protein (PrPC), respectively), have binding sites for Cu and therefore have been suggested to play a role in Cu metabolism. APP possesses two Cu binding domains (CuBD), one located in the N-terminal region between residues 135 and 156 and the other located in the C-terminal region within the amyloid-b peptide (Ab) (Atwood et al., 2000). The CuBD of PrPC is located between residues 60–91 and consists of histidine- and glycine-containing peptide repeats (Stockel et al., 1998). Although the number of octapeptide repeats varies in different species, it is among the most conserved regions of the PrPC in mammals (Wadsworth et al., 1999). In vitro studies in our laboratory determined that the CuBD of both proteins is able to reduce Cu2+ to Cu1+ (Ruiz et al., 1999, 2000; Opazo et al., 2003), suggesting these proteins may normally act as Cu reductases. Cysteine 144 present in the CuBD

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of APP and the tryptophan residues present in the peptide repeats of the CuBD of PrPC are essentials for the reduction process (Ruiz et al., 1999, 2000). Moreover, in vivo studies have revealed that peptides corresponding to the CuBDs of APP and PrPC are able to prevent neurotoxic effects induced by intrahippocampal Cu injection (Chacon et al., 2003; Cerpa et al., 2004). Rats were estereotaxically injected into the dorsal hippocampus with 5 lM CuCl2 in the presence or absence of the wildtype or mutated APP and PrPC peptides. Four days after injection, animals were trained in the Morris Water Maze paradigm over two weeks to evaluate spatial memory acquisition. This paradigm consists of a pool filled with water containing a hidden circular platform where the rats, guided by external cues, are able to escape. The swimming paths at day 8 of training of rats co-injected with Cu plus the CuBDs of the human PrPC (PrP59–91) or APP (APP135–156) were more direct than those of rats injected with Cu alone (Fig. 3A and B, respectively), indicating protection against spatial memory impairment. In contrast to the mutant peptides lacking the

Fig. 3. CuBD of PrPC and APP prevents toxicity induced by intrahippocampal copper injection. Representative swimming paths at day 8 of training of rats injected with copper or copper plus CuBD of PrPC (A) or APP (B). (C) Nissl staining (upper panels, 4· magnification) and GFAP immunodetection (lower panels, 40· magnification) of hippocampal sections of rats injected with aCSF as control, copper, or copper plus the CuBD of APL-1135–156 of C. elegans. Insets in upper panels are shown at 40· magnification. (Adapted from Cerpa et al., 2004).

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copper-binding residues (PrPHis!Ala and APPHis!Ala/His!Ala), co-injection with the mutated peptides that lack residues responsible for Cu reduction (PrPTrp!Ala and APPCys144!Ser) did not prevent the spatial memory loss induced by Cu injection, suggesting that Cu reduction is the main event in the neuroprotective effects of APP135–156 and PrP59–91 (Fig. 3A and B). The behavioral impairment observed was related to morphological changes at the injection site (Table 1). When animals were co-injected with Cu plus APP135–156 or PrP59–91, the neuronal loss and astroglial reactivity was significantly lower that in animals injected with Cu alone. This neuroprotective effect depended on the reduction capacity of the CuBDs, since rats injected with Cu plus APPCys144!Ser and PrPTrp!Ala showed increased neuronal cell loss and astroglyosis compared to rats co-injected with Cu plus the wildtype peptides. We have also studied the neuroprotective properties of the CuBD of C. elegans APP-like protein (APL-1) against Cu2+ neurotoxicity. Rats co-injected with CuCl2 and C. elegans APL-1135–156 showed an escape latency score similar to control rats injected with artificial cerebrospinal fluid (aCSF) (Fig. 3B). Nissl staining of brain sections of rats co-injected with CuCl2 and C. elegans APL-1135–156 showed partial neuroprotection against Cu2+ neurotoxicity (Fig. 3C, upper panel). GFAP immunodetection showed lower astrogliosis and a decrease of hypertrophic astrocytes in rats co-injected with CuCl2 and APL-1135–156 around the injection site, compared to animals injected with CuCl2 alone (Fig. 3C, lower panel). These results suggest that the CuBDs of C. elegans APL-1 and human APP share neuroprotective activities against Cu2+ neurotoxicity, despite their differences in primary structure, and point to a conserved function for the APP fragment throughout evolution. Additional evidence supporting a role for PrPC in Cu homeostasis is the modulation of the PrPC gene (Prnp) expression by copper. Recently, we determined that Cu treatment induces the expression of PrPC in primary hippocampal neurons (Fig. 4A). In addition, Cu induces the activity of a reporter vector driven by the rat Prnp promoter stably transfected into PC12 cells (Varela-Nallar et al., in press). Three different PC12 clones were treated with 100 lM CuCl2 for 16 h. As shown in Fig. 4B, a Table 1 Neuroprotective effect of peptides corresponding to wildtype or mutated CuBDs of human APP and PrPC against intrahippocampal copper injection Treatment

Peptide

Number of neurons at the injection site (±SE)a

Number of reactive astrocytes at the injection site (±SE)b

Control

–

200 ± 6

22 ± 9

CuCl2

– APP135–156 APPCys144!Ser Prp56–91 PrPTrp!Ala

75 ± 8 165 ± 12 95 ± 11 180 ± 11 115 ± 16

48 ± 13 26 ± 12 40 ± 9 32 ± 1 48 ± 6

a The analysis was carry out using Nissl staining and establishing the number of neurons in the superior leaf of the dentate gyrus. b Astroglyosis was analyzed by GFAP immunodetection.

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Fig. 4. Copper induces the expression of PrPC in hippocampal neurons and PC12 cells. (A) Western blot analysis of total cell extracts from hippocampal neurons untreated or treated with 50 lM CuCl2 for 24 h, with polyclonal antibodies against PrPC or b-tubulin. PrP was normalized to b-tubulin levels and expressed as fold-increase over untreated neurons. Bars are mean ± standard error of three independent experiments. (B) PC12 clones stably transfected with a luciferase reporter vector driven by the rat Prnp promoter were treated with 100 lM CuCl2 for 16 h. Luciferase activity of each clone after treatment was expressed as fold-increase over the equivalent untreated clone. Bars are mean ± standard error of three independent experiments. *P < 0.05 according to the single mean StudentÕs-t test. (Adapted from VarelaNallar et al., in press).

large increase in promoter activity, from 20 to 40 times, was observed in all PC12 clones analyzed. The ability of Prnp promoter to respond to Cu is consistent with a role for PrPC in Cu metabolism.

3. Copper and neurodegenerative diseases 3.1. Role of copper in Alzheimer disease Alzheimer disease (AD) is a progressive neurodegenerative disorder that arises on a neuropathological background of amyloid plaques containing amyloid-b-peptide

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(Ab derived from APP and tau-rich neurofibrillary tangles). Ab aggregation is associated with a conformational transition from a-helix to b-plate structure (Soto et al., 1995), which may occur under pathological conditions or as a consequence of aging (Harper and Lansbury, 1997). In vitro analysis showed that Cu did not affect Ab aggregation (Yoshiike et al., 2001) while other studies conclude that it enhance Ab aggregation (House et al., 2004), suggesting that Cu may or may not play a role in the formation of Ab fibrils and in AD pathogenesis. A recent report indicates that, in the presence of copper, there is increased aggregate formation that is associated with the formation of soluble Ab oligomers. These observations are of great interest as it has been established that the oligomers have toxic properties that differ from the soluble monomers and from the fibril aggregates (Kayed et al., 2004). In an in vivo rabbit model for AD, trace amounts of Cu in the drinking water of cholesterol-fed rabbits induced accumulation of Ab formation of senile plaques and retardation of the rabbitÕs ability to learn a difficult task (Sparks and Schreurs, 2003). However, the data on the neuropathological characterization of senile plaques as well as the behavioral studies are presented such that they make a critical assessment of the results difficult. In our laboratory, we have been carrying out studies on transgenic strains of C. elegans that overexpress Ab intracellularly in muscle cells and form Ab aggregates. This model has allowed us to study the aggregation of the Ab peptide in vivo (unpublished results). We have observed that the number and total area of the Ab aggregates are modulated by Cu (Fig. 5). The number of aggregates increases significantly in a time-dependent fashion in worms that have been grown in the presence of the metal (150 lM, 120 h), compared to control animals (Fig. 5 and Table 2). Although these data suggest a noxious role of Cu on Ab aggregation, we observed that Ab overexpression improved survival of worms exposed to increasing Cu concentrations. We found that the worms that express Ab display a lethal concentration 50 (LC50) 37% higher than worms that do not carry the peptide. These data indicate that the presence of Ab confers the worms a greater resistance to environmental copper, suggesting that Ab acts as a Cu chelator and, in this way, diminishes the toxic properties of the metal (Zou et al., 2002). A beneficial effect of Cu on AD pathogenesis has been clearly demonstrated. It was observed that in the transgenic mouse txJ/J (mutant allele of the Cu ATPase7b Cu transporter), Ab species were lower than in age-matched control animals (Phinney et al., 2003). This was manifested by reductions in dense-cored plaques composed of human Ab in APP+/txJ/J mice, including a tendency for reduction of human Ab in the central nervous system (CNS) and in the plasma, and by reduction of endogenous mouse Ab40 and Ab42 in the CNS of young txJ/J mice. On a broader scale, in addition to improving our understanding of AD pathogenesis and risk factors, discerning the mechanism by which txJ can modulate pools of Ab may prove to be of practical use. In addition, Bayer et al. (2003) showed that bio-available Cu is beneficial to transgenic mice over-expressing human full-length APP carrying the Swedish/London mutation (APP23 mice), which is an animal model of AD. Whereas aged APP23 transgenic mice showed a dramatically reduced life expectancy within the observation period, Cu-treated APP23 mice did not show this premature lethal

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Fig. 5. Copper induced aggregation of Ab in a transgenic C. elegans strain. Worms were treated with 150 lm CuCl2 (C and D) or grown under normal conditions (A and B). The figures on the left correspond to 120 h old worms, whereas those on the right to 144 h old animals. The amyloid aggregates (arrows) are visualized with Thioflavine S staining.

Table 2 Copper-modulation of Ab deposits in transgenic C. elegans strain that overexpresses the Ab1–42 peptide Treatment

Number of aggregates (±SE)

Total amyloid deposit area (AU ± SE)

Control Copper

15 ± 1 26 ± 3

700 ± 10 1100 ± 10

phenotype. Moreover, Cu treatment had a modulatory effect on brain Cu levels and a normalizing effect on SOD-1 activity in these mice, compared with non-transgenic littermate control mice. Because the presence of the Cu ion at the active site of human SOD-1 is essential for its enzymatic activity, the authors concluded that the observed rescue of the activity was due to the increasing Cu levels associated with dietary Cu supplementation.

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3.2. Role of copper in prion diseases Prion diseases are fatal transmissible neurodegenerative disorders that include Kuru, Creutzfeld–Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy in cattle (Prusiner, 1998). These diseases are caused by the conformational transition of the native and predominantly a-helical PrPC into a significantly more b-sheet-containing pathogenic isoform (PrPSc) (Pan et al., 1993). Unlike PrPC, PrPSc is insoluble in mild detergents and partially resistant to digestion with proteinase K (PK) (Caughey and Raymond, 1991). The putative role of Cu in prion diseases is not clear and evidence suggests both possible deleterious and beneficial effects. It has been suggested that Cu may induce PrPC misfolding and aggregation since Cu binding induces a conformational change in the unstructured N-terminal region of PrPC, which includes the octarepeat region (Brown et al., 1997; Stockel et al., 1998; Viles et al., 1999; Jackson et al., 2001). Moreover, circular dichroism analysis revealed that Cu binding to a high affinity fifth binding site, outside of the octarepeat region, induces a b-sheet like conformation (Jones et al., 2004). Biochemical evidence also supports a role for Cu in PrPC misfolding. In vitro, Cu converts PrPC extracted from brains of wildtype mice and sheep into a detergent-insoluble and PK-resistant specie (Quaglio et al., 2001). In addition, Cu enhances PK-resistance of PrPSc (Sigurdsson et al., 2003) and facilitates restoration of PK resistance and infectivity of guanidine-denatured PrPSc (McKenzie et al., 1998). It has been observed that Cu induces detergent insoluble PrP in N2a cells (Kiachopoulos et al., 2004), which our group also observed in primary cultured hippocampal neurons. In hippocampal neurons, PrP aggregates show an abnormal glycosylation pattern, suggesting that Cu may induce PrPC misfolding during its biosynthesis and/or intracellular trafficking, leading to accumulation of immature forms (Varela-Nallar et al., in press). The few studies that analyzed the relationship between Cu and prion disease in vivo produced apparently conflicting results. Treatment of scrapie-infected mice with the Cu chelator D-(–)-penicillamine delayed the onset of prion disease, and, at the same time, reduced Cu levels in brain and blood (Sigurdsson et al., 2003), thus favoring the notion that Cu exerts a prion-promoting effect as suggested by in vitro observations. However, an opposite observation is that Cu treatment interferes with the propagation of PrPSc in scrapie-infected N2a cells (Hijazi et al., 2003; Kiachopoulos et al., 2004). Moreover, Cu administration to scrapie-infected hamsters delays the onset of prion disease (Hijazi et al., 2003). These two studies strongly support a beneficial role of Cu against prion disease progression.

4. Concluding remarks As briefly reviewed here, Cu is involved in several cellular events. It is essential to normal cell functioning, but it is also related to the mechanisms of disease. After many years of research, it is still uncertain whether Cu has a beneficial or detrimental effect on the development of neurodegenerative diseases. As described here, evidence

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supports the concept that Cu participates in the aggregation of some proteins and has an effect on the conformation of Ab and PrPC, but the properties of the resulting aggregates have not been completely elucidated and their toxicity is unknown. Moreover, the cellular mechanisms involved in the formation of such aggregates have not been established. In vivo experiments using animal models of both Alzheimer disease and prion diseases support beneficial effects of Cu on the appearance and progression of these diseases. However, there are still many questions to answer before the role of Cu on the development of neurodegenerative diseases can be fully elucidated.

Acknowledgements This research was supported by grants from FONDAP N 13980001, The Millennium Institute for Fundamental and Applied Biology (MIFAB), Millennium Nucleus in Developmental Biology (MNDB) and the International Copper Association (ICA). W. Cerpa and L. Varela-Nallar receive Graduate Fellowships from CONICYT.

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