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The N-terminal copper-binding domain of the amyloid precursor protein protects against Cu²⁺ neurotoxicity in vivo

WALDO F. CERPA^*,1, MARÍA I. BARRÍA^*,1, MARCELO A. CHACÓN^*, MIRIAM SUAZO, MAURICIO GONZÁLEZ, CARLOS OPAZO^*,, ASHLEY I. BUSH^, and NIBALDO C. INESTROSA^*,2

^* Centro FONDAP de Regulación Celular y Patología "Joaquín V. Luco," MIFAB, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, Santiago, Chile;
Instituto de Nutrición y Tecnología de Alimentos, Universidad de Chile, Santiago, Chile;
Mental Health Research Institute of Victoria, Parkville, Parkville, Victoria, Australia; and
Genetics and Aging Research Unit, Harvard Medical School, Massachusetts General Hospital, Charlestown, Massachusetts, USA

²Correspondence: E-mail: ninestr{at}bio.puc.cl

SPECIFIC AIMS

Copper is an essential trace metal, but an unbalance in its levels is associated with oxidative processes (damage to membranes and proteins) that lead to cellular death. The amyloid precursor protein (APP) contains the amyloid ß-peptide, which is involved in the pathogenesis of Alzheimer’s disease. Its physiological function has not been established, but several studies support the notion that APP is involved in Cu homeostasis. This study focuses on the function of a specific APP motif, the copper binding domain (CuBD), located at the N-terminal of this protein, particularly its capacity to protect from copper neurotoxicity in an in vivo rat model.

PRINCIPAL FINDING

1. APP_135-156 protects spatial memory and diminishes neuronal cell loss and astrogliosis induced by copper injection
Human CuBD was used to evaluate its ability to prevent the behavioral impairment caused by the intrahippocampal CuCl₂ injection bilaterally into the dorsal hippocampus (–3.5 mm AP, ±2.0 mm ML, and –2.7 mm DV, according to Bregma). Rats injected with CuCl₂ (5 µM) alone showed spatial memory impairment (Morris Water Maze task), but animals coinjected with 5 µM CuCl₂ and 5 µM of human APP_135-156 incubated and dialyzed behaved like control animals (aCSF-injected rats) in 2 wk of training (Fig. 1 A). The protective effect of human APP_135-156 against Cu²⁺ neurotoxicity was observed with two other protocols of incubation. Therefore, APP_135-156 in the same molar ratio with CuCl₂ protects against Cu²⁺ neurotoxicity.

View larger version (18K): [in this window] [in a new window]   Figure 1. APP135-156 peptides prevent changes on spatial memory induced by Cu2+ neurotoxicity. A) Escape latency scores along 2 wk of training of rats injected with artificial cerebrospinal fluid, aCSF (white circles, n=5), 5 µM CuCl2 (red circles, n=5), and 5 µM CuCl2 + 5 µM of human APP135-156 fragment (blue squares, n=6). B) Representative swimming paths at day 8 of training.

Analyzing hippocampal morphology (Nissl staining), CuCl₂ injection reveals important neuronal loss in the upper leaf of the dentate gyrus compared with APP_135-156 + copper (Fig. 2B vs. C ). Analysis of astrogliosis (derived from GFAP immunohistochemistry) showed that coinjection of human APP_135-156 with CuCl₂ induced only a small astrocytic reaction around the injection site compared with CuCl₂ injection alone.

View larger version (70K): [in this window] [in a new window]   Figure 2. Effect of different CuBD of APP on hippocampal morphology and neuronal loss induced by Cu2+ neurotoxicity. Different CuBD of APP preincubated at 37°C and dialyzed with copper. A) aCSF; B) 5 µM CuCl2; C) 5 µM CuCl2 + 5 µM of human APP135-156; D) 5 µM CuCl2 + 5 µM APPHis147Ala/His149Ala; E) 5 µM CuCl2 + 5 µM APPCys144Ser. Nissl staining: 4x; insets: 40x.

2. Cysteine is a key residue involved in the neuroprotective effects of APP_135-156 against Cu²⁺ neurotoxicity
It has been shown that the N-terminal region of APP is able to bind and reduce Cu(II) to Cu (I). To elucidate which APP biochemical properties were responsible for the neuroprotective effects of APP_135-156, we used synthetic peptides without Cu binding residues (APP_{His147Ala/His149Ala}) or the Cu-reducing residue (APP_Cys144Ser). Rats injected with CuCl₂ + APP_{His147Ala/His149Ala} showed protection of spatial memory as in rats coinjected with CuCl₂ and the wild-type APP_135-156 (Fig. 1B ). Coinjection of CuCl₂ and APP_{His147Ala/His149Ala} induced a lower astrocytic response around the injection site, indicating this peptide is able to decrease neuronal loss in the upper leaf of the dentate gyrus induced by CuCl₂ injection in all the evaluated conditions (Fig. 2 B vs. D). On the other hand, rats coinjected with CuCl₂ and APP_Cys144Ser present higher escape latency values in the memory task close to values observed in rats injected with CuCl₂ alone (Fig. 1A ). The absence of protection by APP_Cys144Ser against Cu²⁺ neurotoxicity was independent of the preincubation protocol used. Nissl staining showed an important neuronal loss in the upper leaf of the dentate gyrus around the injection site in all conditions evaluated (Fig. 2D ). These results suggest that Cu reduction by the CuBD of APP is involved in the neuroprotective effects observed.

3. The CuBD of Caenorhabditis elegans APL-1 protects spatial memory against Cu²⁺ neurotoxicity
To determine whether an APP homologue with variations in the central histidine binding site, but with the adjacent cysteine intact, can protect against Cu²⁺ toxicity, we used the C. elegans CuBD (APP_{135-156C. elegans}). Rats coinjected with CuCl₂ and APP_{135-156 C. elegans} showed an escape latency score similar to control rats independent of the incubation protocol used (data not shown). Immunodetection of GFAP showed a lower astrogliosis induced by CuCl₂ around the injection site in two treatments compared with animals injected with CuCl₂ alone. These results suggest that the CuBD of the C. elegans APP and the CuBD of human APP share neuroprotective activities against Cu²⁺ neurotoxicity, suggesting a conserved function for this APP fragment through out evolution.

4. CuBD of APP prevents protein tyrosine nitration induced by CuCl₂
To determine whether the neuroprotective effect of APP against CuCl₂ neurotoxicity is mediated by an antioxidant pathway, we performed nitrotyrosine immunodetection (-ntyr) as an oxidative marker in the injection site of the animals. The anti-nitrotyrosine staining was carried out 1 wk after the intrahippocampal injection. Results indicate that aCSF injection showed no positive signal whereas CuCl₂ injection induced a clear signal for protein tyrosine nitration. When APP_Cys144Ser was coinjected with CuCl₂, the protein nitration signal was increased. CuCl₂ + APP_{His147Ala/His149Ala} and CuCl₂ + wild-type APP or the APP fragment of C. elegans injections both present low levels of nitrotyrosine protein detection. These data show that the formation of nitrotyrosine proteins is a downstream effect of the Cu²⁺ neurotoxicity that is abolished by the presence of the CuBD of APP via a mechanism that involves Cu(II)/Cu(I) reduction.

5. CuBD of APP increases Cu²⁺ uptake
To determine whether the differences in protection against Cu²⁺ neurotoxicity presented by the CuBD peptides may be related to differences in Cu²⁺ uptake, we coinjected 50 µM ⁶⁴CuSO₄ and 50 µM of APP_135-156, APP_{His147Ala/His149Ala}, APP_Cys144Ser, and APP_{135-156 C.elegans}; 14 h after the injection, Cu²⁺ uptake was determined. Results show a 10-fold increase in Cu²⁺ uptake in the presence of human APP_135-156. In contrast, APP_Cys144Ser exhibited only half the effect of its wild-type counterpart. APP_{His147Ala/His149Ala} and APP_135-156 of C. elegans induced an enhancement in Cu²⁺ uptake. These data are consistent with the idea that APP protects against Cu²⁺ neurotoxicity by inhibiting the formation of reactive oxygen species by direct modulation of the Cu²⁺ levels at the extracellular space.

CONCLUSIONS

The physiological function of APP is unknown, but growing evidence supports the idea that APP may be involved in the Cu homeostasis. APP coordinates Cu forming bioinorganic complexes with high K_d values, suggesting possible in vivo relevance. APP contains in its structure clusters of amino acids ordinarily involved in Cu binding coordination such as histidines and cysteine (see Fig. 3 A, red). CuBD of APP are able to reduce Cu(II) to Cu(I). In this paper, we evaluated whether the CuBD of APP modulates Cu²⁺ neurotoxicity at the level of the hippocampus, performing intrahippocampal injections in rats as an in vivo model. CuCl₂ was injected alone or coinjected with several synthetic peptides corresponding to the CuBD of human wild-type APP, human mutant APP peptides, and APP ortholog of C. elegans.

View larger version (24K): [in this window] [in a new window]   Figure 3. Schematic representations of the effect of CuBD of APP against copper toxicity. A) APP indicating the CuBD sequence, Cu binding coordination of histidine, and cysteine in red. B) Left: Cu uptake by Cu transport proteins. Center: Cu uptake in conditions of high extends copper. Copper interacts with cell membranes causing an increased in oxidative damage. Right: Cu uptake in the presence of the CuBD of APP decreased oxidative stress generated by copper.

Since results may be explained by the need to metallate Cu to the CuBD of APP in order to mediate its effect, the peptides used were metallated. We found no differences between peptides metallated or not, nor any evidence that Cu-metallated CuBD of APP induces neuronal cell death in vivo.

Our studies indicate that when human APP_135-156 was injected + CuCl₂, it protected against Cu²⁺ toxicity at both behavioral and histological levels. To evaluate the role of Cu binding vs. the Cu reduction ability on the CuBD of APP neuroprotective properties, we used CuBD of APP mutated in histidines or cysteine residues (APP_{His147Ala/His149Ala} and APP_Cys144Ser). Coinjection of CuCl₂ + APP_{His147Ala/His149Ala} showed a protection similar to that observed with the APP_135-156 peptide, but when CuCl₂ was coinjected with APP_Cys144Ser there were higher escape latency values in the spatial memory test and increased hippocampal neuronal cell loss and astrogliosis. Our studies indicate that the CuBD of APP protects against the Cu²⁺ neurotoxicity because of its Cu (II)/Cu(I) -reducing capacity.

It was important to evaluate whether copper-induced neurotoxicity was related to oxidative stress. We noted that the injection of CuCl₂ alone or CuCl₂ + APP_Cys144Ser induced a significant immunohistochemical nitrotyrosine signal, indicating that reactive oxidative species were generated inducing oxidative stress. In contrast, coinjection of the human wild-type APP and the APP variant peptide without histidines + CuCl₂ showed only a weak signal.

The protection observed with APP_135-156 agrees with earlier studies demonstrating neuroprotective and neurotrophic activities for soluble APP. In the present work we found increased Cu²⁺ uptake in the presence of the CuBD of APP peptides, which may help decrease the extracellular Cu(I) concentration, which in turn decreases Cu-dependent oxidative stress. For example, APP_Cys144Ser, which did not protect against Cu²⁺ toxicity, showed a Cu uptake capacity 50% less than the wild-type APP_135-156. This agrees with the idea that the intracellular milieu exhibits an overcapacity for Cu chelation; thus, an increase incorporation of Cu may limit the toxicity of the metal in the extracellular milieu.

Cu²⁺/Cu⁺ is an essential micronutrient for humans as it constitutes an important component of various redox enzymes. However, free Cu²⁺/Cu⁺ is a toxic ion, and both excess and deficiency lead to disorders such as Wilson’s and Menkes’s diseases. In eukaryotic cells, incorporation of Cu(II) occurs in a reduced state via the Cu(I) transporter hCtr1.

Therefore, the Cu-reducing activity of APP might serve a favorable physiological function as a cell membrane Cu (II)-reductase similar to Fre1 in yeast, modulating the amount of Cu(I) for subsequent uptake by Cu transport proteins (Fig. 3B , left). In conditions of copper excess (i.e., protocol intrahippocampal injection), copper interacted with cell membranes, causing increased oxidative damage (Fig. 3B , center). The presence of the CuBD of APP allows copper binding and reduction of the copper excess, increasing copper uptake and decreasing oxidative stress generated by damage (Fig. 3B , right). Our data indicate that the CuBD of APP can modulate Cu²⁺/Cu⁺ availability in vivo, supporting the notion that the APP is involved in Cu²⁺/Cu⁺ homeostasis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-1349fje;

¹ These authors contributed equally to this work.

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