Amyloid-ß-peptide reduces copper(II) to copper(I)
independent of its aggregation state

CARLOS OPAZO, FRANCISCA H. RUIZ AND NIBALDO C. INESTROSA¹

Centro de Regulación Celular y Patología. Departamento de Biología Celular y Molecular, ¹Facultad de
Ciencias Biológicas and MIFAB. Pontificia Universidad Católica de Chile

ABSTRACT

Alzheimer’s disease (AD) is characterized by the deposition of amyloid b-peptide (Aß) and neuronal degeneration in brain regions involved in learning and memory. One of the leading etiologic hypotheses regarding AD is the involvement of free radical-mediated oxidative stress in neuronal degeneration. Recent evidence suggests that metals concentrated in amyloid deposits may contribute to the oxidative insults observed in AD-affected brains. We hypothesized that Aß peptide in the presence of copper enhances its neurotoxicity generating free radicals via copper reduction. In the present study, we have examined the effect of the aggregation state of amyloid-ß-peptide on copper reduction. In independent experiments we measured the copper-reducing ability of soluble and fibrillar Aß_1-40 forms by bathocuproine assays. As it was previously observed for the amyloid precursor protein (APP), the Aß peptide showed copper-reducing ability. The capacity of Aß to reduce copper was independent of the aggregation state. Finally, the Aß peptide derived from the human sequence has a greater effect than the Aß peptide derived from the rat sequence, suggesting that histidine 13 may play a role in copper reduction. In agreement with this possibility, the Aß peptide reduces less copper in the presence of exogenous histidine.

Key Words: Aß peptide, amyloid fibrils, copper reduction, oxidative damage, Alzheimer’s Disease.

Abbreviations used: AChE: acetylcholinesterase; Aß: amyloid-ß peptide; AD: Alzheimer’s Disease; APP: amyloid precursor protein; BC: bathocuproine disulfonate; BSA: bovine serum albumin; PBS: phosphate buffered saline.

INTRODUCTION

Alzheimer disease (AD) is characterized pathologically by the presence of neurofibrillary tangles, senile plaques (SP), amyloid-ß-peptide (Aß) deposition, and by a selective loss of neurons. Several lines of evidence found in Alzheimer’s brain (Table 1), as well as markers of oxidative stress in Alzheimer patients, showed a decreased in plasma total antioxidant capacity (TRAP) (Repetto et al., 1999) suggest that oxidative stress is involved in the neurodegeneration observed in AD (Markesbery, 1997; Miranda et al., 2000). However, whereas oxidative damage and antioxidant response are well characterized in AD, the source(s) of damaging reactive oxygen species that trigger such damage is yet to be established. The main component of amyloid deposits is a 4.1-4.3 kDa hydrophobic peptide called amyloid-ß-peptide (Aß) (Soto et al., 1994) that results from the processing of a much larger membrane amyloid precursor protein (APP) (Kang et al., 1987), which possesses metal binding domains (Multhaup et al., 1996).

TABLE I

Oxidative damage markers found in Alzheimer’s Disease Brain
 

Oxidative Stress Markers	Observation	Reference
glucose-6-phosphate dehydrogenase activity	Increased	Martins et al., 1986
heme oxygenase-1 levels	Increased	Smith et al., 1994
free protein carbonyls	Increased	Smith et al., 1996
lipid peroxidation adducts	Increased	Sayre et al., 1997
protein nitration	Increased	Smith et al., 1997
mitochondrial DNA oxidation adducts	Increased	Mecocci et al., 1994
nuclear DNA oxidation adducts	Increased	Mecocci et al., 1994
cytoplasmatic RNA oxidation adducts	Increased	Nunomura et al., 1999

Two lines of evidence connect metals to Alzheimer’s disease. First, metals promote the aggregation of Aß peptide in vitro (Atwood et al., 1998), probably via metal-catalyzed oxidation of the Aß peptide (Dyrks et al., 1992), and second, metals such as copper and iron are accumulated in plaques (Lovell et al., 1998). Interestingly, Aß peptide in a soluble state generates hydrogen peroxide with concomitant TBARS signals, probably by the generation of hydroxyl radical formation (Huang et al., 1999) via Fenton-like or Haber-Weiss reactions (Haber and Weiss, 1934; Halliwell and Gutteridge, 1990). The mechanisms involved in the Aß-mediated neurotoxicity are unknown, although the fibril state of Aß is apparently necessary for the toxicity observed in neuronal cultures (Iversen et al., 1995; Bonnefont et al., 1998; Muñoz and Inestrosa, 1999; Calderón et al., 1999). We hypothetized that the interaction of amyloid fibrils and metals with oxidative properties such as copper may be a source of reactive oxygen species. In the present report we show that the Aß1-40 peptide in both soluble or fibrillar state reduces copper.

METHODS

Synthetic peptides and proteins

Aß_1-40 peptide (human and rat sequence) and APP_135-156 (human sequence) were obtained from Chiron Corp. Inc., Emeryville, CA, USA. The bovine serum albumin (BSA) was obtained from Sigma Chem. St. Louis, U.S.A. Acetylcholinesterase (AChE) was purified from bovine caudate nucleus, as previously described (Inestrosa et al., 1987; Opazo and Inestrosa, 1998).

Copper reduction assay

Copper reduction was analyzed using bathocuproine disulfonate (BC) as a Cu(I) indicador molecule (Multhaup et al., 1996). The Cu(I)-BC complex has a maximum absorbance at 480 nm. For the assay, the samples were incubated in phosphate buffered saline (PBS) at 37 °C for 60 min with 10 µM Cu(II) (copper sulfate), 360 µM BC and various reducing agents. Cu(I) formation was monitored against time as the increases in absorbance at 480 nm.

Aggregation assay: turbidity

The aggregation assay was carried out as described by Alvarez et al., 1997. Specifically, stock solutions were prepared by dissolving lyophilized aliquots of the Aß peptides in dimethyl sulfoxide (DMSO) at 15 mg/ml (3.75 mM). Aliquots of peptide stock (75 nmol in @ 20 µl of DMSO) were added to aqueous buffer (725 µl total vol.; 0.1 M PBS, pH 7.4). Aggregation was measured by turbidity at 400 nm vs. buffer blank. The solutions were stirred continuously (1350 rpm).

Electron microscopy of amyloid fibrils

The amyloid fibrils formed in the turbidity assays were examined by electron microscopy. The fibrils were placed on Formvar-carbon coated 300-mesh nickel grids and negatively stained with 3% phosphotungstic acid solution for one minute. Grids were examined under a Philips EM-300 electron microscope at 60 kV (Inestrosa et al., 1996; Alvarez et al., 1997).

RESULTS

Aß peptide reduces copper (II) to copper(I)

We first analyzed the copper-reducing capacity of the Aß peptide using 10 µM CuCl₂ and compared it to several control compounds. The results presented in Figure 1 indicate that the Aß peptide has the ability to reduce copper, producing approximately 25% of the activity observed using vitamin C, a strong Cu(II) reducing agent. Moreover, this activity was specific for the Aß peptide; other polypeptides like BSA and AChE were not able to induce the same reductive effect. The Aß_1-40 peptide reduced copper, albeit to a lesser extent (35%) than that observed with the APP_135-156 fragment (Ruiz et al., 1999).

Figure 1. Reduction of Cu (II) to Cu (I) by different reducing agents. Samples containing the Cu (I) indicator BC (360 µM), Cu (II) (10 µM) and a reducing agent in PBS were incubated at 37°C for 60 min. The reducing agents used (at 10µg/ml) include: AChE; BSA; Aß, Aß1-40 peptide; APP135-156; Vitamin C. Bars represent the mean ± SEM of 5 different experiments. The reduction of Cu (II) to Cu (I) was quantified by recording the complex formation of Cu(I) with BC at an absorbance maximum of 480 nm.

The reducing activity of Aß1-40 was found to be concentration-dependent in the range 0 to 50 µg/ml of peptide plus 10 µM of copper (Fig. 2A). In order to confirm the involvement of Aß in this effect, the copper-reducing activity of the Aß_1-40 peptide was studied in the presence of vitamin C (0.08 mg/ml) in order to eliminate background activity. As observed in Figure 2B, the DA480 nm was similar to that observed in the absence of ascorbate (Fig. 2A), indicating that the Aß_1-40 peptide is the main factor behind the copper-reducing effect. Similar experiments were performed in presence of histidine Cu-His (1:10) and Aß_1-40 (Fig. 3A). The presence of histidine in the assay decreased the of effect of the Aß peptide in copper reduction, but it did not abolish it completely. As a control experiment, we used a peptide derived from the Aß_1-40 peptide of rat. As Fig 3B shows, the peptide derived from the rat sequence reduced copper to a lesser extent (50%) than did the human derived Aß peptide (Fig 3B).

Figure 2. The Aß1-40 peptide stimulates the reduction of copper (II) to copper (I). (A) 10 µM copper and 360 µM BC were incubated in PBS at 37 °C for 60 min in the presence of Aß peptide at various concentrations (0-50 mg/ml). Cu(I) formation was monitored as the increase in absorbance at 480 nm. (B) Similar experiments were performed in the presence of the reducing agent, vitamin C (0.08 mg/ml). Inset shows the concentration-dependence of copper reduction by vitamin C. Values are means ± SEM of three different experiments.

Figure 3. Effect of histidine on Cu(II) reduction by Aß1-40. A) Copper (10µM) or Cu-His (·10 µM) were incubated in presence of Aß1-40 at various concentrations (0-50 µg/ml) in PBS at 37°C for 60 min. B) Aß1-40 derived from human or rat sequence with copper (10 µM) in PBS at 37°C for 60 min. Cu(I) formation was monitored as the increase in absorbance at 480 nm. Values are means ± SEM of three different experiments.

Amyloid fibrils are also able to reduce copper

We evaluated whether amyloid fibrils formed in vitro from Aß_1-40 possessed copper-reductase activity. Figure 4A shows negatively-stained fibril preparations formed from Aß_1-40 in kinetic stirred aggregation experiments as seen under the electron microscope (Alvarez et al., 1997). Amyloid fibrils, 0-50 µg/ml, were incubated with copper 10 µM at 37oC for 60 min and the reduction of copper was determined (Fig. 4B). Results indicate that the copper-reducing activity of amyloid fibers formed from Aß_1-40 was 1-fold higher than that observed with soluble Aß_1-40 peptide.

Figure 4. Aß1-40 fibrils reduce Cu(II) in a concentration-dependent manner. (A) Electron micrograph of negatively-stained fibril preparations formed from Aß1-40 in a kinetic stirred aggregation experiments. Aliquots of fibril preparations were adsorbed onto 300-mesh Formvar-coated grids, stained with 2% uranyl acetate and viewed for fibrils under the electron microscope. Original magnification 55,000x. Bar represents 0.1 mm. (B) 10 µM copper and 360 µM BC were incubated in PBS at 37°C for 60 min in the presence of either Aß-amyloid fibrils (·), (0-50 mg/ml) or Aß1-40 (), (0-50 mg/ml). Cu(I) formation was monitored as the increase in absorbance at 480 nm. Values are means ± SEM of three different experiments.

DISCUSSION

The Aß peptide, a 40-amino acid peptide involved in the pathology of AD (Selkoe, 1997), showed the ability to reduce copper. One factor probably involved in copper reduction is the presence of two His residues in this sequence, at positions 13 and 14, which seem to play a role in copper binding (Nar et al., 1991; Atwood et al., 1998). In fact, Aß peptide derived from the rat sequence (His13 ® Arg) has 50% less ability to reduce copper than that derived from the human sequence. In agreement with this result, Aß peptide derived from the human sequence presents a minor copper-reducing activity in the presence of exogenous histidine. Previous reports have shown that Aß peptide produces hydrogen peroxide production and a TBARS positive signal (Huang et al., 1999). The hydroxyl radical probably formed in this process may be generated via the Haber-Weiss reaction (Fig. 5) (Haber and Weiss, 1934; Halliwell and Gutteridge, 1990). Free radical-generating systems are able to catalyze the oxidative modification of proteins when Fe(III) or Cu(II) are in the presence of O₂ and an appropriate electron donor (Halliwell and Gutteridge, 1990). In fact, the conversion of superoxide radicals (O₂.) and H₂O₂ to the highly cytotoxic hydroxyl radical (HO.) can only take place when catalytic concentrations of transition metals are present (Valdez et al, 2000). The transition metals accumulated in plaques may be a direct source of reactive oxygen species (Smith et al., 1997) associated with the oxidative stress observed in AD. In fact, transgenic mice that overexpress APP present amyloid deposition associated to redox-active iron and oxidative damage markers (Smith et al., 1998). In accordance with this evidence Lovell et al (1998) found that copper, iron and zinc are concentrated within the core and periphery of plaque deposits. On the other hand, transition metals directly stimulate the aggregation of Aß peptide (Dyrks et al., 1992; Atwood et al., 1998), probably increasing the Aß species that enhance neurotoxicity (Iversen et al., 1995). This evidence indicates that an imbalance of metal ion homeostasis at any level (Nuñez et al., 2000) may have a role in the pathogenesis of AD (Atwood et al., 1998; Sayre et al., 1999).

Figure 5. Hypothetical steps for hydroxyl radical formation in copper reduction by the Aß peptide. (1) Aß peptide reduces copper and generates H2O2 (first step). (2) H2O2 reacts with copper (I) generating hydroxyl radicals via Haber-Weiss reaction (second step). In both step copper may be bound to Aß peptide.

It has previously been suggested that the Aß peptide itself spontaneously generates free radicals that can damage cells (Hensley et al., 1994). Recently however, these results were described as artifactual due to contaminants in some of the preparations (Dikalov et al., 1999). In fact, the Dikalov team showed that the Aß peptide potentiates metal-catalyzed oxidation of hydroxylamines derivatives. In agreement with this result, previous studies showed that the Aß aggregation process was accelerated by transition metals via metal-catalyzed oxidation of the Aß peptide (Dyrks et al., 1992). Interestingly, the Aß peptide produces hydrogen peroxide with its concomitant TBARS signal, probably by way of hydroxyl radical formation (Huang et al., 1999). Moreover, we found that amyloid fibrils reduce copper, suggesting that oxygen radical species can be generated in the initial and final steps of the amyloid formation process. APP also reduces copper (II) to copper (I) (Fig. 1) (Multhaup et al., 1996; Ruiz et al., 1999), and subsequent exposure to hydrogen peroxide results in the re-oxidation of Cu(I) and site-specific cleavage of APP (Multhaup et al., 1998). In a cellular context, the favorable or deleterious effects of the copper-reducing activities of Aß and APP will depend upon the extent of APP expression and Aß amyloid accumulation. Trace metal elements are necessary for normal cell function (Zago et al., 2000), and in this context the ability of APP and the Aß peptide to reduce copper should serve a favorable physiological function, possibly presenting Cu(I) to the Cu(I) to the membrane transporter (Zhou and Gitschier, 1997). Under unfavorable conditions, an abnormal increase of APP, or during an accumulation of Aß peptide into amyloid fibrils, copper may be further reduced, thereby generating an increase in Cu(I) levels, H₂O₂ and free radicals with the consequent oxidative damage (Multhaup et al., 1998; Ruiz et al., 1999), such as lipid peroxidation (Cadenas and Sies, 1998) or protein oxidation, a process which may alter the protein function (Hidalgo et al., 2000) or increase protein degradation (Davies et al., 1987).

ACKNOWLEDGEMENTS

This work was supported by a Pre-Doctoral Fellowship from CONICYT to C.O., and FONDECYT grant Nº 2990087 to C.O., CIMM-ICA/006 to N.C.I., and FONDAP grant N° 13980001. N.C.I is the recipient of a Presidential Chair in Science from the Chilean Government (1999-2001). The Millenium Institute of Fundamental and Applied Biology was funded in part by the Chilean Ministry of Planning and Development.

Corresponding Author: Dr. N.C. Inestrosa Departamento de Biología Celular y Molecular, Unidad de Neurobiología Molecular , P. Universidad Católica de Chile. Casilla 114-D, Santiago, Chile. Fax: (56-2) 686-2717. e-mail: ninestr@genes.bio.puc.cl
¹ Address correspondence and reprint requests to at.

Received: January 3, 2000. Accepted: January 3, 2000

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