IUBMB
Life, 57(9): 645 – 650, September 2005
Critical Review Copper Brain Homeostasis: Role of Amyloid Precursor Protein and Prion Protein Nibaldo C. Inestrosa, Waldo Cerpa and Lorena Varela-Nallar Centro de Regulacion Celular y Patologı´a ‘‘Joaquin V. Luco’’ (CRCP), MIFAB, Facultad de Ciencias Biolo´gicas, Pontificia Universidad Cato´lica de Chile, Santiago, Chile
Summary The main proteins associated with Alzheimer’s and prion diseases (amyloid precursor protein (APP) and prion protein (PrPC), respectively, have binding sites for copper and it has therefore been suggested that they play a role in copper metabolism. Here, we review evidence indicating that the copper binding domains (CuBD) of APP and PrPC are able to modulate the oxidation state of copper, and prevent neurotoxic effects and memory impairments induced by copper. Results with transgenic and other animal models have established the relation between these pathogenic proteins and copper. In particular, APP transgenic models, suggest a beneficial effect for copper in AD. IUBMB Life, 57: 645 – 650, 2005 Keywords
Copper; APP; prion protein; metal homeostasis; copper binding domain.
APP AND ITS ROLE IN COPPER HOMEOSTASIS The amyloid precursor protein (APP) is a cell surface transmembrane glycoprotein containing the amyloid-b peptide (Ab), which is involved in the pathogenesis of Alzheimer’s disease (AD) (1). APP possesses two copper binding domains (CuBD): one located in its N-terminal region between residues 135 and 156 and the other located in the C-terminal region within the Ab domain (2) (Fig. 1), suggesting that they may have a functional link between APP and copper. In fact, APP knockout mice show increased copper levels in cerebral cortex, in contrast with APP-overexpressing transgenic mice (3), supporting the concept that APP should participate in copper metabolism. In addition, APP CuBD reduces Cu+2 to Cu+1 (4), and we determined that cysteine 144 present in the CuBD is essential for this process (5) (Table 1). Received 28 February 2005; accepted 12 July 2005 Address correspondence to: Dr Nibaldo C. Inestrosa, CRCP Biomedical Center, P. Catholic University of Chile, Alameda 340, Santiago, Chile. Fax: +56 2 6862959. E-mail:
[email protected] ISSN 1521-6543 print/ISSN 1521-6551 online Ó 2005 IUBMB DOI: 10.1080/15216540500264620
We carried out in vivo studies with synthetic peptides corresponding to the CuBD of the human APP (APP135 – 156) to evaluate its ability to prevent the behavioral impairment caused by the intrahippocampal injection of copper (6). As observed in the representative swimming paths at day 8 of training, rats injected with CuCl2 alone showed spatial memory impairments (Fig. 2A); however, animals co-injected with CuCl2 plus human APP135-156 behaved like control animals (aCSF injected rats) after two weeks of training. This neuroprotection was also observed histologically when neuronal cell loss and astrogliosis were examined (6). These results indicate that human APP135 – 156 protects from Cu2+-induced neurotoxic effects. To elucidate which of the two APP biochemical properties, binding or reduction of Cu2+ to Cu1+ (4, 5), were responsible for the neuroprotective effects of APP135 – 156, three mutant peptides were synthesized bearing specific amino acid substitutions: two at the His-X-His-X-His sequence located between amino acids 147 and 151, and the other at the only cysteine present in the APP135 – 156 fragment (Table 1). Rats injected with CuCl2 plus peptides without the Cu-binding residues (APPHis147!Ala/His149!Ala) showed protection against spatial memory impairment (Fig. 2A), as also occurs with rats co-injected with CuCl2 and the wild-type APP135 – 156. Rats injected with CuCl2 plus the peptide without the Cu-reducing residue (APPCys144!Ser) show spatial memory close to rats injected with CuCl2 alone. An important point to evaluate was whether the copperinduced neurotoxicity was related to oxidative stress; analysis of 3-nitrotyrosine residues indicated increasing levels of nitrotyrosine in AD (7). We have observed that copper neurotoxicity correlated with an increase in nitrotyrosine immunofluorescence and a decrease in Cu2+-uptake (6). The injection of CuCl2 alone or CuCl2 plus APPCys144!Ser (Fig. 3) induced an important increase in nitrotyrosine signal, indicating the generation of reactive oxidative species. In contrast,
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co-injection of the human wild-type APP135 – 156 or the APPHis147!Ala/His149!Ala variant peptide plus CuCl2 only showed a weak signal (Fig. 3). The protection exerted by APP135–156 is in agreement with previous studies demonstrating neuroprotective and neurotrophic activities for soluble APP (8, 9). In conclusion, the evidence indicate that the CuBD of APP can modulate Cu2+/Cu1+ availability in vivo, supporting the notion that the APP is involved in Cu2+/Cu1+ homeostasis (5, 10). Figure 1. Schematic representation of copper binding domain (CuBD) of amyloid precursor protein (APP) and prion protein (PrP).
Figure 2. APP135 – 156 PrP59 – 91 peptides prevent changes on spatial memory induced by Cu neurotoxicity. Representative swimming paths at day 8 of training of rats injected with copper in the absence or presence of APP and PrP peptides. (A) The neuroprotective effect of 5 mM APP135 – 156 and APPHis!Ala/His!Ala against copper toxicity is clearly shown. APPCys!Ser shows only a partial protective effect. (B) PrP59 – 91 and PrPHis!Ala at 5 mM prevents memory impairments induced by copper. This neuroprotection is not observed by PrPTrp!Ala.
THE PATHOLOGICAL ROLE OF COPPER IN ALZHEIMER’S DISEASE Besides the suggested relation between APP and copper at a functional level, there is also evidence that suggests that copper may be related to AD pathogenesis. The interaction between copper and Ab induces its aggregation in vitro (11) and favours Ab amyloid deposition in vivo, co-localizing in senile plaques; in fact, copper, iron and zinc levels increase in plaques up to 0.4 mM copper and up to 1 mM iron and zinc (12). Treatment with a copper-zinc chelator markedly and rapidly inhibits amyloid deposition in the brain of Alzheimer’s disease transgenic mice (13). The relationship between copper, APP and the Ab levels has been recently studied. In fact, it has been shown that Ab plus metal generates H2O2 utilizing O2 and biological reducing agents as substrates. The Km indicates that this enzyme-like reaction is likely to occur under physiological conditions. Ab must therefore generate a specific structure that presents redox-active Cu to O2 and certain reducing substrates (14). Ab also may act as a metal chelator (15). Sparks et al. (2003) (16) showed that trace amounts of copper in the drinking water of cholesterol-fed rabbits induces accumulation of Ab, formation of senile plaques, reduction of glutathione peroxidase activity, increases in SOD activity and retardation of the rabbit’s ability to learn a difficult task. They suggest that cholesterol entering the brain from the circulation of cholesterol-fed rabbits induces the neuronal accumulation
Table 1 Effect of CuBD of APP and PrP in copper reduction in vitro APP135-156 Wt His147!Ala His147!Ala His149!Ala Cys144!Ser
% Cu1+ formation
Prp59-91
% Cu1+ formation
100 + 5 70 + 4
Wt
100 + 8
58 + 2 7+1
Trp ! Ala
19 + 4
Cu1+ was monitored by the formation of a peak with maximal absorbance at 480 nm in the presence of bathocuproine disulfonic acid. Table shows the increase in the percentage with respect to wild-type CuBD.
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Figure 3. Effect of different CuBD of APP and Cu in the nitrotyrosine signal in the rat hippocampi. Nitrotyrosine immunofluorescence of coronal brain sections of rats injected with: (A) aCSF; (B) 5 mM CuCl2; (C), 5 mM CuCl2 plus 5 mM of human APP135 – 156; (D) 5 mM CuCl2 plus 5 mM APPHis147!Ala/His149!Ala; (E) 5 mM CuCl2 plus 5 mM APPCys144!Ser.
of Ab, and that copper influences the clearance of Ab from the brain, at the level of the blood brain barrier. Squitti et al. (17) presented evidence indicating that elevated serum levels of Cu and ceruloplasmine are found in AD patients, however, the serum increases did not indicate directly what happens in neurons. More importantly, Bayer et al. (18) showed that bioavailable copper is beneficial to transgenic mice over-expressing human full-length APP with the Swedish mutation (APP23 mice). Whereas aged APP23 transgenic mice showed a dramatically reduced life expectancy within the observation period, Cu-treated APP23 mice did not show this premature death phenotype. Moreover, Cu treatment had a modulating effect on brain Cu levels and a normalizing effect on SOD-1 activity in these mice, compared with non-transgenic littermate controls. 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 copper levels on dietary Cu supplementation. Finally, they stated that their ‘observation should be regarded as a proof-of-concept for a prophylactic approach to address a nervous system copper deficiency in AD’. Phinney et al. (2003) (19) showed that copper levels in brains of APP-overexpressing transgenic TgCRND8 mice were lower than in non-Tg controls, even though the Tg mice exhibit a substantial burden of dense-cored plaques and high Ab levels. Moreover, Ab species were lower in txJ/J mice (mutant of the ATPase7b copper transporter favoring elevated copper levels) than in age-matched controls. This was showed by a reduction in dense-cored plaques composed of human Ab in APP+/txJ/J mice, including a tendency for reduction of human Ab in the brain and in the plasma, and by reduction of endogenous mouse Ab 40 and Ab42 in young txJ/J mice. More broadly, besides improving our understanding of Alzheimer pathogenesis and the risk factors, discerning the mechanism whereby txJ can modulate pools of Ab may prove to be of practical use. The above in vivo evidence strongly suggests a beneficial effect for Cu in different AD models (18, 19), indicating that Cu could be a positive player in stopping the progress of AD.
PRION PROTEIN AND ITS PHYSIOLOGICAL ROLE IN COPPER HOMEOSTASIS Prion diseases are caused by the conformational transition of the predominantly a-helical PrPC into a significantly more b-sheet enriched pathogenic isoform (PrPSc) (20), which is accompanied by changes in the biochemical properties of the protein. While PrPC is soluble in mild detergents and sensitive to proteinase K (PK) digestion, PrPSc is insoluble in mild detergents and shows partial resistance to PK digestion (21). PrPC is a cell surface N-linked glycoprotein mainly expressed in neurons (22) attached to the cell surface by a glycosylphophatidylinositol (GPI)-anchor (23). The physiological function of PrPC is still unknown, but several lines of evidence suggest its role in copper metabolism. PrPC binds copper ions with low micromolar affinity via histidine and glycine-containing peptide repeats in its Nterminal region (24, 25) (Fig. 1). This copper binding domain is located between residues 60 – 91 and consists of four identical repeats of the peptide sequence Pro-His-Gly-GlyGly-Trp-Gly-Gln. Although the number of octapeptide repeats varies in different species, it is among the most highly conserved region of the PrPC in mammals (26), suggesting it plays a role in PrPC function. The octarepeat region is highly selective for Cu2+ and the binding of the metal is pHdependent (24). Besides copper binding, it was determined in our laboratory that the octarepeat region of PrPC also bears the capacity of reduce copper in vitro (27) (Table 1). The incubation of Cu+2 with a peptide corresponding to the octarepeat region of the human PrPC (PrP59 – 91) resulted in the reduction of Cu+2 to Cu+1. This reduction depends on the tryptophan residues present in the octapeptide repeats since a mutant peptide of the octarepeat region lacking the tryptophan residues (PrPTrp!Ala) showed a dramatic decrease in Cu+2 reduction with respect to the wild-type peptide (27) (Table 1). These observations suggest that PrPC may act as a copper reductase facilitating its incorporation into the cell. These results have been recently confirmed and extended (28). We have observed that the octarepeat region of PrPC protects against copper neurotoxicity in vivo, in fact, we showed that neuronal cell loss and astrogliosis induced by
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intrahippocampal injection of copper were reduced by coinjection with the peptide PrP59 – 91 (29). In addition, coinjection with PrP59 – 91 prevented behavioural impairments caused by intrahippocampal copper injection which was evaluated in the Morris water maze. Injection of 5 mM copper induced high levels of escape latency compared to cerebrospinal fluid injection even after several days of training (Fig. 2B). Co-injection of 5 mM copper plus 5 mM PrP59 – 91 decreased escape latency values indicating protection against spatial memory impairment. The neuroprotective effect of PrP59 – 91 can be clearly observed in Fig. 2B, which shows representative swimming paths at day 8 of training. We determined that the neuroprotective effect of PrP59 – 91 is mediated by the tryptophan residues as observed in rats co-injected with copper plus a PrP59 – 91 peptide lacking the tryptophan residues (PrPTrp!Ala) (Fig. 2B). As observed in the swimming paths, this mutant peptide did not prevent the spatial memory loss induced by copper injection. In contrast, a mutant PrP59 – 91 peptide lacking the copper-binding residues (PrPHis!Ala) did prevent spatial memory impairments induced by copper (Fig. 2B). These results indicate that instead of the copper binding ability, copper reduction is the main event in the neuroprotective effects of PrP59 – 91. Although the binding of copper to PrPC have a number of consequences biologically, the physiological relevance of this binding is unknown. Copper binding has an effect on PrPC trafficking, inducing the endocytosis of PrPC from the cell surface in a reversible manner (30), suggesting that PrPC may act as a receptor for cellular uptake or efflux of copper. However, a more recent study showed that while PrPC expression increases copper binding at the cell surface it does not result in increased intracellular copper concentration (31). Moreover, we have recently observed in primary cultured hippocampal neurons and PC12 cells that copper treatment increases the expression of the PrPC gene (32), suggesting a functional link between copper and PrPC. While all the above mentioned evidence suggests a link between the physiological function of PrPC and copper metabolism, the specific function of PrPC remains elusive.
COPPER AND PRION DISEASES Several studies have focused on the effect of copper on PrP conformation and it has been suggested that copper might play a role in PrPC misfolding. In vitro, copper binding to PrPC induces a conformational change in the unstructured N-terminal region of the protein (25). Recently, it has been determined by circular dichroism that copper binds to a fifth binding site of high affinity (Fig. 1), outside of the octarepeat region, inducing b-sheet like conformation (33). Biochemical evidence also supports a role for copper in PrPC misfolding. In vitro, copper converts PrPC extracted from brains of wild-type mouse and ovine into a detergent insoluble and PK-resistant species (34, 35). Copper also enhances PK resistance of PrPSc (36) and facilitates
restoration of PK resistance and infectivity of guanidinedenatured PrPSc (37). This evidence suggests that copper may promote conversion of PrPC into PrPSc. Moreover, copper induces changes in PrPC detergentsolubility in N2a cells (38) suggestive of PrPC misfolding. We also observed that copper treatment induces detergent insoluble PrPC aggregates in primary cultured hippocampal neurons (39). Copper-induced PrP aggregates of both hippocampal neurons and N2a cells show a different glycosylation pattern than PrPC in control cells, and show similarities to the pattern of glycosylation associated with the scrapie-like conversion induced by the proteasome inhibition (40). Although in vitro evidence suggest that copper may participate in the conformational alteration of PrPC related to prion diseases, in vivo observations have been controversial. Treatment of scrapie-infected mice with the copper chelator D-(– )-penicillamine delayed the onset of prion disease (36), supporting the notion that copper exerts a prion promoting effect. However, copper administration to scrapie-infected hamsters delays the onset of prion disease (41), suggesting a beneficial role of copper against prion disease progression. In conclusion, previous observations clearly illustrate the controversy that still exists regarding the role of copper in Alzheimer and prion diseases. After several years of research the role of this metal in the pathogenesis of these neurodegenerative diseases is beginning to be clarified with the evidence favoring a beneficial effect for copper.
ACKNOWLEDGEMENTS This work was supported by grants from FONDAP-Biomedicine N813980001, The Millennium Institute for Fundamental and Applied Biology (MIFAB) and the International Copper Association (ICA)-New York.
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