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Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
J Neurochem. Author manuscript; available in PMC 2014 July 14.
Published in final edited form as:
PMCID: PMC4096128

Chronic copper exposure exacerbates both amyloid and tau pathology and selectively dysregulates cdk5 in a mouse model of AD


Excess copper exposure is thought to be linked to the development of Alzheimer disease (AD) neuropathology. However, the mechanism by which copper affects the central nervous system remains unclear. To investigate the effect of chronic copper exposure on both beta-amyloid and tau pathologies, we treated young triple transgenic (3xTg-AD) mice with 250 ppm copper-containing water for the period of 3 or 9 months. Copper exposure resulted in altered APP processing; increased accumulation of the amyloid precursor protein (APP) and its proteolytic product, C99 fragment, along with increased generation of amyloid-beta peptides and oligomers. These changes were found to be mediated via upregulation of BACE1 as significant increases in BACE1 levels and deposits were detected around plaques in mice following copper exposure. Furthermore, tau pathology within hippocampal neurons was exacerbated in copper-exposed 3xTg-AD group. Increased tau phosphorylation was closely correlated with aberrant cdk5/p25 activation, suggesting a role for this kinase in the development of copper-induced tau pathology. Taken together, our data suggest that chronic copper exposure accelerates not only amyloid pathology but also tau pathology in a mouse model of AD.


Alzheimer disease (AD), a leading cause of dementia among the elderly, is characterized by the presence of senile plaques and neurofibrillary tangles composed of amyloid-beta (Aβ) and hyperphosphorylated tau, respecitively. Approximately, 5% of people over the age of 65 develop AD, and this number is progressively increasing over time. To date, the etiopathogenesis of idiopathic AD remains unkown. However, epidemiological studies suggest that environmental factors may play an important role in the pathogenesis of the disease, either as a trigger or as a modulator of disease progression. Among them, heavy metal exposures potentially modulate AD pathology and have impact on amyloidogenesis. Copper is one of the heavy metals that has a strong binding affinity to amyloid precursor protein (APP) and Aβ, and it has been hypothesized that the presence of copper may facilitate the production as well as aggregation of Aβ in the brain (Atwood et al. 1998; Bush 2003; Tougu et al. 2008). In support for a role of metal ions in AD, post-mortem studies revealed significantly elevated levels of heavy metals including copper, iron, and zinc in human AD brain as compared with agematched controls (Lovell et al. 1998; Bush 2003), and these metals were highly localized to senile plaques (Lovell et al. 1998). Furthermore, studies using animal models of AD found that chronic copper intake exacerbated Aβ pathology and impaired cognitive function (Sparks and Schreurs 2003; Lu et al. 2006). On the other hand, a study found that elevating brain copper levels stabilized superoxide dismutase-1 (SOD1) and lowered amyloid burden in a transgenic mouse model of AD (Bayer et al. 2003). Therefore, due to divergent findings, the effects of increased copper on Aβ remain unclear.

In the current study, we examined the effects of chronic copper exposure on Aβ and tau pathologies in 3xTg-AD mice. The potential influence of copper on tau-related pathology has not been previously examined, and thus this is the first study to examine the effect of copper on both plaques and tangles in the same model. We found that chronic copper exposure in young 3xTg-AD mice lead to significant alterations in APP processing, including increased steady-state levels of APP and C99 and enhanced production of Aβ and oligomeric species. The upregulation of BACE1 may mediate this change, and the increased BACE1 deposits around plaques were also associated with numbers of plaques in the brain. Furthermore, we found that tau phosphorylation was significantly exacerbated as detected by increased phosphorylation at ser202/thr205 (AT8), thr231/ser235 (AT180) and ser396/ser404 (PHF-1). Marked increase of p25 fragment along with increased calpain activity was detected in chronic copper-exposed mice, indicating that copper triggered aberrant activation of cdk5 and tau phosphorylation is closely correlated with cdk5/p25 activation. Thus, our findings suggest that prolonged exposure to excessive copper leads not only to elevations in Aβ but also enhances the development of tau-related neuropathology.


Animals and treatment paradigm

2-month old 3xTg-AD mice (Thy1.2-APPswe, Thy1.2-TauP301L, PS1M146V-KI) were treated with 250 ppm copper sulfate (CuSO4) in the drinking water for a period of 3 or 9 months. The drinking water contained 5% sucrose to enhance intake. Control groups were given 5% sucrose containing drinking water for the same period. Each group consisted of an equal number of males and females, and the total number of mice was 10 per group.


Primary antibodies used in this study are summarized in Table 1. Secondary biotinylated antibodies (anti-mouse, anti-rat and anti-rabbit), normal sera (Vector Laboratories, Burlingame, CA), and secondary antibodies for immunofluorescent staining (AlexaFluor 488, 546 or 568 for anti-mouse and anti-rabbit; Molecular Probes, Eugene, OR) were also used in this study.

Immunohistochemical staining for APP/Aβ, tau, and activated microglia (CD45) were conducted as previously described (Oddo et al. 2003; Kitazawa et al. 2005). For immunostaining for oxidative markers, free-floating sections (50 µm thickness) were pretreated with Tris buffered saline (TBS) containing 3% hydrogen peroxide and 10% methanol for 30 min to block endogenous peroxidase activity. After a TBS wash, sections were incubated once in TBS with 0.1% Triton X-100 (TBST) for 15 min and once with TBST with 2% BSA for 30 min. Sections were incubated with the primary antibody (see Table 1) in TBS + 5% normal goat or horse serum overnight at 4°C. Sections were then incubated with secondary antibody (biotinylated anti-rabbit or antimouse; 1:200 in TBS + 2% BSA + 5% normal serum) for 1 hr at room temperature, followed by Vector ABC and DAB (Vector Laboratories) to visualize the staining.

For double immunofluorescent staining with thioflavin S (Sigma, St. Louis, MO) and microglia, free-floating sections were incubated with 0.5% thioflavin S in 50% ethanol for 10 min. Sections were washed twice with 50% ethanol for 5 min each, once with water for 5 min, once with TBST for 15 min and once with TBST with 2% BSA for 30 min. Staining was visualized using a BioRad 2100 confocal microscope.


Brains were homogenized in T-PER (Pierce, Rockford, IL) in the presence of a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN) and phosphatase inhibitors (5 mM sodium fluoride and 50 µM sodium orthovanadate), and centrifuged at 100,000 xg for 1 hr at 4°C. Supernatants were collected as the detergent-soluble fraction. Pellets were resuspended in 70% formic acid, homogenized and centrifuged at 100,000 xg for 1 hr at 4°C. Resultant supernatants were collected as the detergent-insoluble or formic acid (FA) fraction. These fractions were immunoblotted with antibodies that recognize APP, C99, total tau, phosphorylated tau, cdk5, p35/p25, GSK-3β, phospho-GSK-3β, p38-MAPK, phospho-p38-MAPK, JNK or phospho-JNK. Membranes were reprobed with antibody against µ2;-actin to control for protein loading. Band intensity was measured using Quantity One software (BioRad Laboratories, Hercules, CA).


Copper chaperone for SOD1 (CCS) was immunoprecipitated from all brain lysates. 150 µg of proteins were incubated with 2 µg of anti-CCS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) overnight at 4°C with gentle rocking. Protein A-agarose plus (Calbiochem) was added and further incubated for 2 hrs at 4°C. Protein-antibody complex was isolated by serial centrifugation and washing, and immunoprecipitated proteins were subsequently analyzed by immunoblotting using Clean-Blot IP detection reagent (Pierce).

Dot blot analysis for oligomeric Aβ

Brain homogenates (5 µg) were placed onto the nitrocellulose membrane and airdried for 20 min. The membrane was then blocked with 5% fat-free milk for 2 hrs and incubated with primary antibody A11 (1:2000) overnight at 4°C. Signals were detected by secondary anti-rabbit-HRP conjugated antibody and Supersignal Dura detection reagent (Pierce).

ELISA for Aβ40 and Aβ42

40 and Aβ42 were detected in both the detergent-soluble and -insoluble fractions by enzyme-linked immunosorbent assay (ELISA). Soluble fractions were loaded directly onto ELISA plates and FA fractions were diluted 1:20 in neutralization buffer (1 M Tris base; 0.5 M Na2HPO) prior to loading. MaxiSorp immunoplates (Nunc) were coated with monoclonal Aβ20.1 antibody at a concentration of 25 µg/ml in coating buffer (0.1 M NaCO3 buffer, pH 9.6), and blocked with 3% BSA. Synthetic Aβ standards were defibrillated by dissolving in HFIP at 1 mg/ml and the HFIP evaporated with a stream of nitrogen. The defibrillated Aβ was then dissolved in DMSO at 1 mg/ml. Standards of both Aβ40 and Aβ42 were made in antigen capture buffer (ACB; 20 mM NaH2PO4; 2 mM EDTA, 0.4 M NaCl; 0.5 g CHAPS; 1% BSA, pH 7.0), and loaded onto ELISA plates in duplicate. Samples were loaded in duplicate and incubated overnight at 4°C. Plates were washed and then probed with either HRP-conjugated anti-Aβ35–40 (MM32-13.1.1, for Aβ1–40) or anti-Aβ35–42 (MM40-21.3.4, for Aβ1–42) overnight at 4°C. 3,3’,5,5’-tetramethylbenzidine was used as the chromagen, and the reaction stopped by 30% O-phosphoric acid, and read at 450 nm on a Molecular Dynamics plate reader.

Calpain activity assay

Brains were homogenized with T-PER without protease inhibitors. Following the standard extraction procedures described above, detergent-soluble fractions (2 µg/µl) were used to determine calpain activity by InnoZyme calpain activity assay kit (Calbiochem). The endogenous calpain activity was quantified by reading the fluorescence at an excitation of 340/20 nm and an emission of 480/20 nm using Synergy HT fluorescent plate reader (Bio-Tek Instruments, Winooski, VT).

Cell culture and quantitative RT-PCR

Human neuroblastoma SHSY-5Y cells were maintained in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 50 units penicillin and 50 µg/ml streptomycin at 37°C in a humidified atmosphere containing 5% CO2. Cells were plated in 6-well culture plate at a density of 250,000 cells/well and incubated for 24 hrs. Cells were then exposed to copper containing media at a concentration ranging from 10 µM (2.5 ppm) to 1 mM (250 ppm) for 24 hrs. Detergent-soluble fractions were collected using M-PER (Pierce) in the presence of protease and phosphatase inhibitors for immunoblot analysis. RNA was extracted using Aurum total RNA mini kit (Bio-Rad Laboratories) for RT-PCR.

100 ng of RNA was subsequently used for one-cycle reverse transcriptase reaction to make cDNA using iScript cDNA synthesis kit (Bio-Rad Laboratories) and was subjected to real-time PCR to quantify expressions of APP using iQ SYBR Green supermix (Bio-Rad Laboratories). The following primers were used for the reaction: huamn APP (forward) 5’-GAC AAA TAT CAA GAC GGA GGA-3’, (reverse) 5’-CCA CAC CAT GAT GAA TGG ATG TG-3’; mouse APP (forward) 5’-GGG GCC GCA AGC AGT GCA AG-3’, (reverse) 5’-CCC CAC CAG ACA TCA GAG T-3’; actin (forward) 5’-ACT GTG TTG GCA TAG AGG TCT TTA-3’, (reverse) 5’-CTA GAC TTC GAG CAG GAG ATG G-3’ (Green et al. 2006). Cycle of threshold (Ct) was calculated by MyiQ software (Bio-Rad Laboratories), and the quantitative fold changes in mRNA were determined as relative to actin mRNA levels.

Quantitative and statistical analyses

All immunoblot data were quantitatively analyzed using Bio-Rad Quantity One software or Image J software. Statistics were carried out using one-way ANOVA with post-tests or unpaired t-test, and p<0.05 or lower was considered to be significant.


3-month copper exposure results in the accumulation of APP and selectively increases levels of Aβ40 in the brain

To study the chronic effects of copper exposure in AD-related pathology, 2 months old 3xTg-AD mice were exposed to 250 ppm copper in the drinking water for a period of 3 or 9 months. During the exposure period, body weight was measured weekly, and no significant group differences in animal weight were detected (data not shown). Thus, the level of copper utilized did not cause any obvious systemic toxicity, consistent with previous reports using the same concentration of copper in another mouse model (Bayer et al. 2003).

Copper exposure for a period of 3 months resulted in a significant elevation of the steady-state levels of APP in the brain (Fig. 1A, B). Similarly, the levels of C99, but not C83, were also markedly increased in the copper-exposed group (Fig. 1A, C), indicating overall upregulation of APP processing and related pathology. Likewise, BACE1 levels were also upregulated in the brain following copper exposure, whereas no apparent difference was detected in ADAM10 or PS1 levels (Fig. 1A, D), indicating that the selective aberration of C99 could be mediated via copper-induced upregulation of BACE1 activity. The increase in mRNA levels of APP and BACE1 levels following copper exposure has been recently reported in PC12 cells (Lin et al. 2008). To further explore the mechanism of copper-induced upregulation of APP observed in the mouse model, we exposed copper to human neuroblastoma SH-SY5Y cells for 24 hrs. The steady-state levels of APP were dose-dependently increased in the range of 10 to 100 µM (2.5 to 25 ppm) copper exposures (Suppl. Fig. 1A). On the other hand, we did not find a significant increase in endogenous APP mRNA levels by RT-PCR following the copper exposure (Suppl. Fig. 1B), suggesting post-translational processes may be altered and involved in the accumulation of APP in copper-exposed SH-SY5Y cells. The exposure to 1 mM (250 ppm) copper was highly toxic to cells and resulted in a significant reduction of the steady-state levels of APP (Suppl. Fig. 1A).

Figure 1
3-month copper exposure alters APP processing

ELISA demonstrated that selective increase of Aβ40 was detected in copper-exposed mice while no alteration of production/accumulation of Aβ42 was observed between the groups (Fig. 1E). Although the total Aβ load in the brain was increased following copper exposure, no extraneuronal plaques (as detected by 6E10 antibody or thioflavin S) were evident in either group at 6 month old 3xTg-AD mice (Fig. 1F).

We next examined whether tau pathology was exacerbated by copper exposure. The steady-state levels of total tau were not altered between controls and copper-exposed mice as measured using antibody HT7 (Fig. 2A). This finding suggests that copper did not alter Thy1.2 transcription activity. Likewise, phospho-tau levels were not found to be altered between the groups by immunoblots or immunostaining (Fig. 2A, B). However, thorough analysis of stained sections revealed that the number of AT8-positive neurons in CA1 region of the hippocampus of copper-exposed mice was significantly more than control group (Fig. 2B), suggesting that copper-induced change in tau pathology became just evident at 3 months of exposure in 3xTg-AD mice.

Figure 2
3-month copper exposure does not exhibit pathological tau accumulation

9-month copper exposure exacerbated both Aβ and tau pathologies in the 3xTg-AD mice

Based on the results from the 3-month copper exposure, we next examined the effects of copper exposure for a longer period (9 months) in the 3xTg-AD mice. The steady-state levels of APP remained statistically higher in the copper-exposed group (Fig. 3A, B). However, the expressions of both endogenous mouse APP and human APP transgene were not significantly altered by copper (Suppl. Fig. 1C). The downstream APP processing resulted in marked elevation of C99 as well as C83 levels as compared to the control group (p<0.05; Fig. 3A, C). This alteration of APP processing appeared to be accompanied by a robust upregulation of BACE1 levels in the brain of copper-exposed mice (Fig. 3A, D). Unlike the 3 month copper exposure data, ADAM10 levels were also significantly increased following 9-months of copper exposure, which resulted in the elevation of the non-amyloidogenic component, C83 fragment. The increased production of Aβ levels was further confirmed by ELISA, and soluble Aβ40 levels remained significantly higher in the copper-treated group (Fig. 3E). In the detergent-insoluble fraction, however, the significance was lost due to high variability among individual mice although Aβ40 levels tended to be higher in the copper-exposed group (Fig. 3F). Immunohistochemical staining revealed that Aβ-containing plaques were present in the subicular region of the hippocampus in both groups, and these extraneuronal plaques were immunoreactive to both Aβ40 and Aβ42 (Fig. 3G). Increased BACE1 levels were detected in brain homogenates of copper-exposed mice (Fig. 3A, D). A recent study demonstrated that increased BACE1 deposition was observed in the vicinity of amyloid plaques in a transgenic mouse model, suggesting an important role in development of plaques in the brain (Zhao et al. 2007). We determined that increased BACE1 depositions were also evident in copper-exposed mice (Suppl. Fig. 2). BACE1 was predominantly found around plaques.

Figure 3
9-month copper exposure exacerbates Aβ pathology in the brain

In addition, oligomeric species were also detected around the plaques in both groups (Fig. 4A). Quantitative measurement of oligomers revealed that copper-exposed mice had relatively increased amount of A11-positive oligomers in the brain although it failed to show a statistical significance (Fig. 4A). Oligomers in the brain were particularly found in surroundings of the amyloid plaques, which may be the evidence that oligomers were precursors for the maturation of plaques. Interestingly, A11-positive astrocytic cells were also stained around the plaques, and double labeling with GFAP confirmed A11-positive oligomer-containing astrocytes in the brain (Fig. 4B), suggesting that oligomers may be ingested by these monocyte cells as previously described (Parvathy et al. 2008).

Figure 4
Detection of oligomeric Aβ species in the brain

We next examined changes in tau pathology following 9-month copper exposure. Significant increases in AT8-, AT180- and PHF-1-positive phosphorylated tau were evident in the copper exposed animals as compared to controls (Fig. 5A). Again, total tau levels as detected by HT7 did not differ between treatment groups, suggesting that 9 months of copper exposure in 3xTg-AD mice resulted in increased tau hyperphosphorylation without exerting an effect on transgene expression levels (Fig. 5A). Consistent with immunoblot analysis, immunohistochemical staining showed somatodendritic accumulations of AT8-positive tau were increased in the hippocampus as well as late-stage pathological tau detected by PHF-1 antibody were also more apparent in the copper-exposed group as compared to the control group (Fig. 5B). These data clearly show that chronic copper exposure exacerbates both plaques and tangle pathologies in the brain.

Figure 5
9-month copper exposure exacerbates tau pathology

Exacerbation of tau pathology correlates with aberrant cdk5/p25 activation

To further elucidate the cellular mechanisms of copper-induced pathological tau phosphorylations in the 3xTg-AD mice, we examined activations of cyclin-dependent kinase 5 (cdk5) and glycogen synthase kinase-3β (GSK-3β), two major kinases associated with abnormal tau phosphorylation in the brain. The aberrant activation of cdk5 can be detected by measuring p35/p25 levels, co-activators of cdk5, and increased formation of the p25 fragment has been suggested to promote pathological tau formation in AD as well as in mouse models of the disease (Patrick et al. 1999; Lee et al. 2000; Augustinack et al. 2002; Cruz et al. 2003). Likewise, GSK-3β activation is regulated by phosphorylation at serine 9, and active GSK-3β has been reported to phosphorylate tau and facilitate tangle formation (Ferrer et al. 2002; Noble et al. 2005; Leroy et al. 2007). Following 3 months of copper exposure, no apparent changes were detected in the steady-state levels of cdk5, p35/p25, GSK-3β or phospho-GSK-3β (Fig. 6A, B, Suppl. Fig. 3). This is consistent with our findings that phospho-tau levels were not elevated at this time point (Fig. 2). On the other hand, exacerbation of tau pathology following 9-months of copper exposure in 3xTg-AD mice was accompanied by increased formation of p25, a cytosolic activator of cdk5, while the activation status of GSK-3β remained insignificant between the groups (Fig. 6A, B, Suppl. Fig. 3). The cytosolic p25 is generated through the proteolytic cleavage of p35 by calpain activity. We further determined whether brain calpain activity is increased in 9-month copper exposure in the 3xTg-AD mice. In the copper-exposed brain, calpain activity was generally higher than control mice although it failed to show a statistical significance due to high variability among the mice (Fig. 6C)‥ Collectively, our result indicates that copper-mediated increase of phospho-tau levels closely correlated with the activation of cdk5/p25 in the brain.

Figure 6
Copper exposure activates cdk5 via increased generation of p25

Copper exposure alters protein interaction with CCS

Chronic copper exposure to young 3xTg-AD mice favored amyloidogenic process of APP cleavage in the brain as measured by increasing levels of C99 and Aβ. This change was accompanied with increased steady-state levels and deposition of BACE1 in the brain from copper-exposed mice as described above. Recent studies indicate that BACE1 has a copper-binding site in its cytosolic domain and was also found to interact with copper chaperone for superoxide dismutase-1 (CCS), an important protein that transports copper to SOD1 for its activation (Angeletti et al. 2005). To examine whether alteration of CCS binding in BACE1 took place in the brain during chronic copper exposure and modulated BACE1 as well as SOD1 activities, we sought to evaluate the interactions of these proteins. While steady-state levels of BACE1 were upregulated in both 3- and 9-month copper exposed groups (Fig. 1 and and3),3), CCS and SOD1 levels were unchanged in the brain of these mice (Fig. 7). CCS was immunoprecipitated from brain samples, and interaction of BACE1 or SOD1 with CCS was determined by immunoblotting. At 3-month copper exposure, the interaction of BACE1 with CCS significantly increased, whereas the interaction of SOD1 was significantly decreased (Fig. 7). On the other hand, at 9-month copper exposure, the reduction of SOD1 interaction remained, but the increased interaction of CCS-BACE1 was abolished (Fig. 7). As we also observed an increased oxidative stress in copper-exposed brain (Suppl. Fig. 4), our findings may partially explain the underlying mechanisms involved in a decreased SOD1 activity and increased BACE1 activity following copper exposure, and its effect on amyloidgenic pathology in the mouse model.

Figure 7
Brain interaction of BACE1 or SOD1 with CCS is altered in chronic copper exposure


The effects of copper on AD pathogenesis are controversial as some animal studies show beneficial, while others detrimental, (Lee et al. 2002; Bayer et al. 2003; Sparks and Schreurs 2003). The same concentration of copper (250 ppm) was used in the previous study and shown to significantly increase the brain copper concentration, reduce Aβ burden and stabilize SOD1 activity in the brain of transgenic mouse model (Bayer et al. 2003). In human, a recent epidemiological study uncovered a significant association of copper intake with cognitive decline in individuals with high fat diet (Morris et al. 2006). However, it is still an open argument whether dietary or excess copper intake triggers altered APP processing and subsequent development of clinical AD pathologies.

Our present study provides additional evidence showing that chronic copper exposure may be a risk factor for AD. In the 3xTg-AD mouse model, copper exposure not only increases Aβ generation, particularly Aβ40, but also triggers pathological tau phosphorylation and tangle formation in the brain. We demonstrate that increased Aβ generation may be mediated by the upregulation of APP and BACE1 levels in the brain, and that the exacerbation of tau pathology correlates with the increased formation of p25 and subsequent aberrant activation of cdk5/p25. It is, however, not well understood how copper in the brain triggers these pathological changes.

To date, APP is known to possess a copper binding site in its N-terminal extracellular domain (Barnham et al. 2003; Valensin et al. 2004) and has been speculated to regulate copper homeostasis in the brain (White et al. 1999a; Maynard et al. 2002). Copper binding to APP appears to promote dimerization of APP and may further facilitate the localization into lipid rafts where BACE1 and the γ-secretase complex are concentrated, resulting in increased APP processing and the generation of Aβ peptides (Kawarabayashi et al. 2004). Although the down-stream effect/process of APP-copper binding is not well characterized, the concentration of copper in the brain seems to be a critical factor for activating pathological processes.

Increased brain copper concentration also regulates the expression of APP. In vitro studies have reported that chronic copper exposure to cells markedly upregulates APP and BACE1 mRNA levels at relevant concentrations we used in our study (Lin et al. 2008) as well as an even lower concentration for a longer period (Armendariz et al. 2004). The copper-induced altered APP-associated gene expression is one potential mechanism of dysregulation of APP processing in the brain. However, in our study, no significant increase of APP expression in mouse model and SH-SY5Y cells was found following copper exposure, while the steady-state levels of APP significantly elevated. In the 3xTg-AD mouse model, both APP and tau expressions are driven by Thy1.2 promoter, but only the steady-state levels of APP, but not tau, were significantly increased after 3- and 9-month copper exposures. Therefore, we think other mechanisms than altered gene expression may be involved in the increased accumulation of APP by copper exposure. It is critical to further explore the underlying mechanisms of copper on APP expression and post-translational modifications including stabilization and degradation.

Not only APP but also Aβ peptides are also capable of binding to copper as well as iron and zinc, and high levels of these metals (as high as 0.4 mM of copper, for example) are found in senile plaques of AD (Lovell et al. 1998). Upon forming a complex with these metals, Aβ peptides utilize them as a seed and initiate the aggregation process (Atwood et al. 1998; Atwood et al. 2000). An in vitro study demonstrated that Aβ aggregation depends on metal concentration (Hutchinson et al. 2005). Several animal studies demonstrated enhanced aggregation of Aβ and plaque formation in the brain following copper or zinc exposure (Lee et al. 2002; Sparks and Schreurs 2003). Similarly, we observed elevated brain Aβ, particularly Aβ40 in copper-exposed 3xTg-AD mice. At this point, it is not clear how selective generation of Aβ40 occurred in our mouse model.

On the other hand, copper has also been shown to act as a beneficial agent for AD-related pathology. For example, in vitro study demonstrates that copper-treated CHO cells exhibit inhibition of the amyloidogenic pathway via activating the non-amyloidogenic α-secretase activity (Borchardt et al. 1999) as well as in vivo study using a transgenic mouse model of AD with mutant ATPase7b copper transporter results in the buildup of intracellular copper significantly reducing amyloid burden in the brain (Phinney et al. 2003). Another study indicated that the effect of clioquinol on attenuating APP/Aβ pathology is mediated by altering the re-distribution of copper, which facilitates copper uptake into the cell, rather than by the chelating activity of copper from the system (Cherny et al. 2001; Treiber et al. 2004). However, a recent study suggests a potent toxicity of clioquinol mediated by copper transport in the APP transgenic mouse model (Schafer et al. 2007). In accord, our in vivo results show increased α-secretase (ADAM10) and non-amyloidogenic cleavage of APP as detected by increased C83 fragment following chronic copper exposure. However, we also observed an increased BACE1 levels at an even earlier period and subsequent activation of the amyloidogenic pathway, suggesting that the beneficial process does not become evident through the copper exposure in 3xTg-AD mice.

In addition to the direct effect of copper on APP/Aβ, copper exposure also facilitates the generation of reactive oxygen species through mechanisms involved in redox processes. Increased oxidative stress is one of the pathological features of AD as well as other neurodegenerative disorders, and copper may mediate this reaction to contribute to AD pathogenesis. To support this hypothesis, primary neurons with wild-type APP expression were found to be more susceptible to copper toxicity and produced significantly higher levels of oxidative stress than neurons from APP knock-out mice (White et al. 1999b). The increased oxidative stress following copper exposure may be relevant to the expression of BACE1 levels in neurons. Recent studies demonstrated that BACE1 has a copper-binding site in its cytosolic domain, and BACE1 was also found to interact with CCS, an important protein that transports copper to SOD1 for its activation (Angeletti et al. 2005). Since BACE1 competes with SOD1 for the interaction with CCS, upregulation of BACE1 results in the reduced activity of SOD1 in the cells. We found upregulation of BACE1 levels in copper-exposed 3xTg-AD brain along with increasing C99 levels. Furthermore, we detected increased oxidative stress in brains with upregulated BACE1 following chronic copper exposure, in support of BACE1 competing with SOD1, thus leading to oxidative damage.

In conclusion, chronic copper exposure exacerbates brain AD-like pathology via multiple mechanisms. Thus, dyshomeostasis of brain copper levels may be one of the triggering factors of pathogenesis of AD.

Supplementary Material

Suppl Fig 1

Copper exposure increases the steady-state levels of APP in SH-SY5Y cells:

(A) The steady-state levels of APP increase dose-dependently following a 24-hr copper exposure (0.01 – 1 mM or 2.5 – 250 ppm) in SH-SY5Y cells. Densitometric analysis of APP band shows a significant increase (*p<0.05) in 0.1 mM copper exposure and a significant decrease (*p<0.05) in 1 mM copper exposure. Graph represents mean ± S.E.M. of three separate experiments in triplicate. (B) Total RNA extraction and RT-PCR are performed following copper exposure in SH-SY5Y cells. The quality of extracted RNA is confirmed by the presence of 18S and 28S of ribosomal RNA (top right). APP mRNA levels are quantitatively measured by one-step reverse transcriptase reaction followed by RT-PCR (top left). Graph represents the fold difference of APP mRNA levels normalized by actin mRNA (mean ± S.E.M. of three separate experiments in triplicate). No significant difference is detected. Representative RT-PCR reaction cycle is shown in the bottom right. Light green baseline is a negative control (no RNA sample). Bottom right graph represents RT-PCR reaction cycle of selected samples without reverse transcriptase reaction step, confirming no DNA contamination in the samples. (C) 100 ng of total RNA extracted from brains of 9-month copper-exposed or control 3xTg-AD mice were subjected to run RT-PCR for human APP, mouse APP and actin. Data are expressed as mean fold increase ± S.E.M. (n=4 for control and n=6 for copper-exposed mice). No significant difference was observed in both human (transgene) and mouse (endogenous) APP following copper exposure.

Suppl Fig 2

BACE1 deposition around plaques increases following chronic copper exposure:

Brain sections were triple stained with thioflavin S (green), BACE1 (red) and nuclei (blue). Increased plaques as well as BACE1 depositions were detected in copper-exposed 3xTg-AD mice (as shown in the graph below, *p<0.05 compared to control, n=5 per group). Higher magnification image from copper-exposed mice is shown in the last panel.

Suppl Fig 3

Densitometric analysis of steady-state levels of p35, cdk5 and GSK-3β following copper expsure in 3xTg-AD mice:

Densitometric analysis of immunoblots shown in Figure 6. No difference is observed (n=10 per group).

Suppl Fig 4

Chronic copper exposure increases oxidative stress in the brain:

Selected oxidative markers were examined in 9-month copper exposure in 3xTg-AD mice. (A, C) Malonaldehyde (MDA) levels are detected in the CA1 region of hippocampus of the control and copper-exposed mice. The intensity of cytosolic MDA is relatively higher in copper-exposed mice (n=5 per group). (B, D) DNA/RNA oxidative marker, 8-oxo-2-deoxyguanosine (8oxodG), is detected in the subicular region of hippocampus of the control and copper-exposed mice. Significantly more neurons are immunostained with 8oxodG antibody in the copper-exposed mice than the control (p<0.05 compared to control, n=5 per group).


This work was partly supported by a grant from the Alzheimer's Association grant and by the National Institutes of Health grants AG17968 (F.M.L.), AG0212982 (F.M.L.), K99AR054695 (M.K.). Aβ antibodies were provided by the UCI Alzheimer's Disease Research Center (ADRC) funded by NIH/NIA grant P50AG16573 and the Institute for Brain Aging and Dementia (IBAD) funded by the NIH Program Project Grant, AG00538.


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