Chronic copper exposure exacerbates both ... - Wiley Online Library

JOURNAL OF NEUROCHEMISTRY

| 2009 | 108 | 1550–1560

doi: 10.1111/j.1471-4159.2009.05901.x

Department of Neurobiology and Behavior, University of California, Irvine, California, USA

Abstract Excess copper exposure is thought to be linked to the development of Alzheimer’s disease (AD) neuropathology. However, the mechanism by which copper affects the CNS remains unclear. To investigate the effect of chronic copper exposure on both beta-amyloid and tau pathologies, we treated young triple transgenic (3·Tg-AD) mice with 250 ppm copper-containing water for a period of 3 or 9 months. Copper exposure resulted in altered amyloid precursor protein processing; increased accumulation of the amyloid precursor protein and its proteolytic product, C99 fragment, along with increased generation of amyloid-beta peptides and oligomers. These changes were found to be mediated via up-regulation

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 3·Tg-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. Keywords: Alzheimer, kinase, metal, oligomer. J. Neurochem. (2009) 108, 1550–1560.

Alzheimer’s disease (AD), a leading cause of dementia among the elderly, is characterized by the presence of senile plaques and neurofibrillary tangles composed of amyloidbeta (Ab) and hyperphosphorylated tau, respectively. Approximately, 10% 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 triggers or as modulators 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 Ab, and it has been hypothesized that the presence of copper may facilitate the production as well as aggregation of Ab 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 age-matched 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 Ab 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, as a result of divergent findings, the effects of increased copper on Ab remain unclear.

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Received September 29, 2008; revised manuscript received December 23, 2008; accepted January 12, 2009. Address correspondence and reprint requests to Masashi Kitazawa, Department of Neurobiology and Behavior, 1216 Gillespie Neuroscience Research Facility, University of California, Irvine, CA 92697-4545, USA. E-mail: [email protected] Abbreviations used: Ab, amyloid-beta; AD, Alzheimer’s disease; APP, amyloid precursor protein; BSA, bovine serum albumin; CCS, copper chaperone for SOD1; GSK-3b, glycogen synthase kinase-3b; SOD1, superoxide dismutase-1; TBS, Tris buffered saline.

2009 The Authors Journal Compilation 2009 International Society for Neurochemistry, J. Neurochem. (2009) 108, 1550–1560

Copper exposure accelerates amyloid and tau pathology | 1551

In the current study, we examined the effects of chronic copper exposure on Ab and tau pathologies in 3·Tg-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 3·Tg-AD mice lead to significant alterations in APP processing, including increased steady-state levels of APP and C99 and enhanced production of Ab and oligomeric species. The up-regulation 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 Ab but also enhances the development of taurelated neuropathology.

Materials and methods Animals and treatment paradigm Two-month-old 3·Tg-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. Immunohistochemistry Primary antibodies used in this study are summarized in Table 1. Secondary biotinylated antibodies (anti-mouse, anti-rat and antirabbit), normal sera (Vector Laboratories, Burlingame, CA, USA), and secondary antibodies for immunofluorescent staining (AlexaFluor 488, 546 or 568 for anti-mouse and anti-rabbit; Molecular Probes, Eugene, OR, USA) were also used in this study. Immunohistochemical staining for APP/Ab, 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 lm thickness) were pre-treated 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

Table 1 Antibodies used in this study Antibody

Immunogen

Host

Application

Source

6E10 APP CT20 Ab40 Ab42 A11 HT7 AT8 AT180 PHF-1 CD45 Cdk5 p35/p25 GSK-3b Phospho-GSK-3b BACE1 BACE1 (3D5) ADAM10 PS1 CCS SOD1 MDA 8oxodG Actin

aa 1–17 of Ab aa 751–770 of human APP aa 35–40 of Ab aa 35–42 of Ab Conformation specific oligomers aa 159–163 of human tau Peptide containing phospho-S202/T205 Peptide containing phospho-T231/S235 Peptide containing phospho-S396/S404 CD45 from mouse B-cell aa 268–283 of human cdk5 C-terminus of human p35 aa 1–160 of rat GSK-3b Phosphoepitopes around ser9 of human GSK-3b aa 485–501 of human BACE1

Mouse Rabbit Mouse Mouse Rabbit Mouse Mouse Mouse Mouse Rat Rabbit Rabbit Mouse Rabbit Rabbit Mouse Rabbit Rabbit Mouse Rabbit Rabbit Mouse Rabbit

WB, IHC WB ICH, ELISA ICH, ELISA IF WB, IHC WB, IHC IHC IHC IHC WB WB WB WB WB IF WB WB IP/IB IP/IB IHC IHC WB

Pierce Calbiochem Drs. Cribbs and Vasilevko Drs. Cribbs and Vasilevko Dr. Glabe Pierce Pierce Pierce Dr. P. Davis Serotec Calbiochem Santa Cruz Biotechnology BD Transduction Laboratories Cell Signaling Calbiochem Dr. Vassar Calbiochem Cell Signaling Santa Cruz Biotechnology Novus Biologicals Affinity Bioreagents QED Biosciences Sigma-Aldrich

aa 735–749 of mouse ADAM10 Epitopes around va1293 of human PS1 aa 1–274 of human CCS Full length SOD1 Malonaldehyde deoxy guanosine adduct 8-hydroxyguanosine-BSA and -casein conjugate: C-terminal actin fragment (C11)

Calbiochem, San Diego, CA, USA; Pierce, Rockford, IL, USA; Serotec, Rayleigh, NC, USA; Santa Cruz Biotechnology, Santa Cruz, CA, USA; BD Transduction Laboratories, Lexington, KY, USA; Cell Signaling Technology, Beverly, MA, USA; Novus Biologicals, Littleton, CO, USA; Affinity Bioreagents, Golden, CO, USA; QED Biosciences; Sigma-Aldrich, St. Louis, MO, USA. aa, amino acid; WB, western blot; IHC, immunohistochemistry.


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TBST with 2% bovine serum albumin (BSA) for 30 min. Sections were incubated with the primary antibody (see Table 1) in TBS + 5% normal goat or horse serum overnight at 4C. Sections were then incubated with secondary antibody (biotinylated antirabbit or anti-mouse; 1 : 200 in TBS + 2% BSA + 5% normal serum) for 1 h at 25C, followed by Vector ABC and DAB (Vector Laboratories) to visualize the staining. For double immunofluorescent staining with thioflavin S (Sigma, St. Louis, MO, USA) 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 Bio-Rad 2100 confocal microscope. Immunoblotting Brains were homogenized in T-PER (Pierce, Rockford, IL, USA) in the presence of a protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN, USA) and phosphatase inhibitors (5 mM sodium fluoride and 50 lM sodium orthovanadate), and centrifuged at 100 000 g for 1 h at 4C. Supernatants were collected as the detergent-soluble fraction. Pellets were resuspended in 70% formic acid, homogenized and centrifuged at 100 000 g for 1 h at 4C. Resultant supernatants were collected as the detergentinsoluble or formic acid fraction. These fractions were immunoblotted with antibodies that recognize APP, C99, total tau, phosphorylated tau, cdk5, p35/p25, glycogen synthase kinase-3b (GSK-3b), phospho-GSK-3b, p38-MAPK, phospho-p38-MAPK, c-Jun N-terminal kinase (JNK) or phospho-JNK. Membranes were re-probed with antibody against b-actin to control for protein loading. Band intensity was measured using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Immunoprecipitation Copper chaperone for SOD1 (CCS) was immunoprecipitated from all brain lysates. 150 lg of proteins were incubated with 2 lg of anti-CCS antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA) overnight at 4C with gentle rocking. Protein A-agarose plus (Calbiochem, San Diego, CA, USA) was added and further incubated for 2 h at 4C. 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 Ab Brain homogenates (5 lg) were placed onto the nitrocellulose membrane and air-dried for 20 min. The membrane was then blocked with 5% fat-free milk for 2 h and incubated with primary antibody A11 (1 : 2000) overnight at 4C. Signals were detected by secondary anti-rabbit-HRP conjugated antibody and Supersignal Dura detection reagent (Pierce). Enzyme-linked immunosorbent assay for Ab40 and Ab42 Ab40 and Ab42 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 formic acid 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 Ab20.1 antibody at a concentration of 25 lg/mL in coating buffer (0.1 M NaCO3 buffer, pH 9.6), and blocked with 3% BSA. Synthetic Ab standards were defibrillated by dissolving in HFIP at 1 mg/mL and the HFIP evaporated with a stream of nitrogen. The defibrillated Ab was then dissolved in DMSO at 1 mg/mL. Standards of both Ab40 and Ab42 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 4C. Plates were washed and then probed with either HRP-conjugated anti-Ab35-40 (MM32-13.1.1, for Ab1-40) or anti-Ab35-42 (MM40-21.3.4, for Ab142) overnight at 4C. 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 lg/lL) 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, USA). Cell culture and quantitative RT-PCR Human neuroblastoma SH-SY5Y cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 50 units penicillin and 50 lg/mL streptomycin at 37C 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 h. Cells were then exposed to copper containing media at a concentration ranging from 10 lM (2.5 ppm) to 1 mM (250 ppm) for 24 h. 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. One hundred nanograms of RNA was subsequently used for onecycle 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¢-GACAAATATCAAGACGGAGGA-3¢, (reverse) 5¢-CCACACCATGATGAATGGATGTG-3¢; mouse APP (forward) 5¢-GGGGCCGCAAGCAGTGCAAG-3¢, (reverse) 5¢-CCCCACCAGACATCAGAGT-3¢; actin (forward) 5¢-ACTGTGTTGGCATAGAGGTCTTTA-3¢, (reverse) 5¢-CTAGACTTCGAGCAGGAGATGG-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.



Results Three-month copper exposure results in the accumulation of APP and selectively increases levels of Ab40 in the brain To study the chronic effects of copper exposure in ADrelated pathology, 2-month-old 3·Tg-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 and b). Similarly, the levels of C99, but not C83, were also markedly increased in the copper-exposed group (Fig. 1a and c), indicating overall up-regulation of APP processing and related pathology. Likewise, BACE1 levels were also up-regulated in the brain following copper exposure, whereas no apparent difference was detected in ADAM10 or PS1 levels (Fig. 1a and d), indicating that the selective aberration of C99 could be mediated via copperinduced up-regulation 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 (a)

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up-regulation of APP observed in the mouse model, we exposed copper to human neuroblastoma SH-SY5Y cells for 24 h. The steady-state levels of APP were dose-dependently increased in the range of 10–100 lM (2.5–25 ppm) copper exposures (Fig. S1a). On the other hand, we did not find a significant increase in endogenous APP mRNA levels by RTPCR following the copper exposure (Fig. S1b), 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 (Fig. S1a). Enzyme-linked immunosorbent assay demonstrated that selective increase of Ab40 was detected in copper-exposed mice while no alteration in production/accumulation of Ab42 was observed between the groups (Fig. 1e). Although the total Ab 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 3·Tg-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 and b). However, thorough analysis of

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Fig. 1 Three-month copper exposure alters APP processing. (a) Immunoblot analysis of APP, C99, C83, BACE1, ADAM10 (full-length, 100 kDa) and PS1 in brain homogenates from control or copperexposed mice (n = 10 per group). b-Actin levels are used as a loading control. Densitometric analyses of steady-state levels of (b) APP, (c) C99 and C83, and (d) BACE1 and ADAM10 are presented in graphs

with mean ± SEM (*p < 0.05 compared to control group). (e) Total Ab levels from detergent-soluble and -insoluble fractions are measured by ELISA (n = 10 per groups). *p < 0.05 compared to control group. (f) Representative immunohistochemical staining of APP/Ab by 6E10 antibody.



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(b)

Fig. 2 Three-month copper exposure does not exhibit pathological tau accumulation. (a) Immunoblot analysis of total tau (HT7) and various phospho-tau epitopes including pS202/pT205 tau (AT8), pS396/ pS404 tau (PHF-1) and pT231/pS235 tau (AT180) in brain homogenates from control or copper-exposed mice (n = 10 per group). bactin levels are used as a loading control. Densitometric analyses are

shown in the graph (mean ± SEM). No significant differences of total tau or phospho-tau levels are detected between control and copperexposed mice. (b) Representative immunohistochemical staining of tau in hippocampus region. AT8-positive neurons in CA1 hippocampus are counted and and determined the significance in the graph shown on the right panel (n = 10 per group, *p < 0.05 compared to control).

stained sections revealed that the number of AT8-positive neurons in CA1 region of the hippocampus of copperexposed 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 3·Tg-AD mice.

were not significantly altered by copper (Fig. S1c). 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 and c). This alteration of APP processing appeared to be accompanied by a robust up-regulation of BACE1 levels in the brain of copper-exposed mice (Fig. 3a and 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 Ab levels was further confirmed by ELISA, and soluble Ab40 levels remained significantly higher in the copper-treated group (Fig. 3e). In the detergent-insoluble fraction, however, the significance was lost because of high variability among individual mice although Ab40 levels

Nine-month copper exposure exacerbated both Ab and tau pathologies in the 3·Tg-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 3·Tg-AD mice. The steady-state levels of APP remained statistically higher in the copperexposed group (Fig. 3a and b). However, the expressions of both endogenous mouse APP and human APP transgene



(a) Fig. 3 Nine-month copper exposure exacerbates Ab pathology in the brain. (a) Immunoblot analysis of APP, C99, C83, BACE1, ADAM10 (full-length, 100 kDa) and PS1 in brain homogenates from control or copper-exposed group (n = 10 per group). b-Actin levels are used as a loading control. Densitometric analyses of steadystate levels of (b) APP, (c) C99 and C83, and (d) BACE1 and ADAM10 are presented in graph (*p < 0.05 or **p < 0.01 compared to control group). Total Ab levels from (e) detergent-soluble and (f) detergent-insoluble fractions are measured by ELISA (n = 10 per groups). *p < 0.05 compared to control group. (g) Representative immunohistochemical staining of APP/Ab by 6E10, Ab40 and Ab42 specific antibodies.

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tended to be higher in the copper-exposed group (Fig. 3f). Immunohistochemical staining revealed that Ab-containing plaques were present in the subicular region of the hippocampus in both groups, and these extraneuronal plaques were immunoreactive to both Ab40 and Ab42 (Fig. 3g). Increased BACE1 levels were detected in brain homogenates of copper-exposed mice (Fig. 3a and 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 (Fig. S2), as BACE1 was predominantly found around plaques. 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

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Fig. 4 Detection of oligomeric Ab species in the brain. (a) Representative double immunofluorescent staining of thioflavin Spositive amyloid plaques and A11-positive amyloid oligomers in the brains of control and 9-month copper-exposed mice. Dot blot analysis of A11-positive oligomers demosntrates a marginal increase in copperexposed mice (n = 6 per group). (b) Representative double immunofluorescent staining of astrocytic marker, GFAP, and A11-positive amyloid oligomers in the brain of 3·Tg-AD mice. Arrowheads represent colocalization of GFAP and A11-positive staining, indicating A11-positive oligomers are present in the astrocytes.

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Fig. 5 Nine-month copper exposure exacerbates tau pathology. (a) Immunoblot analysis of total tau (HT7) and phospho-tau epitopes including pS202/pT205 tau (AT8), pS396/pS404 tau (PHF-1) and pT231/pS235 tau (AT180) in brain homogenates from control or copper-exposed mice (n = 10 per group). b-Actin levels are used as a loading control. Densitometric analyses reveal significant increases in brain phospho-tau levels in copper-exposed mice (mean ± SEM, *p < 0.05 or **p < 0.01 compared to control). (b) Representative

immunohistochemical staining of tau in hippocampus region. AT8positive neurons in CA1 hippocampus are counted and determined the significance in the graph shown on the right panel (n = 10 per group, **p < 0.01 compared to control). The arrowheads in PHF-1 staining show highly condensed somatodendritic accumulation of phospho-tau, suggestive of late pathological tau aggregates in CA1 hippocampus of copper-exposed mice.

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). We next examined changes in tau pathology following 9month 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 3·Tg-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 plaque and tangle pathologies in the brain. Exacerbation of tau pathology correlates with aberrant cdk5/p25 activation To further elucidate the cellular mechanisms of copperinduced pathological tau phosphorylations in the 3·Tg-AD mice, we examined activations of cyclin-dependent kinase 5 (cdk5) and GSK-3b, 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-3b activation is regulated by phosphorylation at serine 9, and active GSK-3b



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Fig. 6 Copper exposure activates cdk5 via increased generation of p25. (a) Cdk5 and GSK-3b activation states were determined by immunoblotting of cdk5, p35/p25, phospho-GSK-3b (at serine 9), and total GSK-3b in 3-month and 9-month copper-exposed group and control group (n = 10 per group). (b) Densitometric analysis of p25 fragment is shown in the graph (mean ± SEM, **p < 0.01 compared to age-matched control). A significant increase in the formation of p25 is detected in 9-month copper-exposed mice. (c) Brain calpain activity of 9month copper-exposed mice is measured using a specific fluorogenic substrate. The calpain activity is expressed as relative fluorescent unit (R.F.U.), and the graph represents mean ± SEM of control (n = 4) and copper-exposed mice (n = 6).

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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-3b or phospho-GSK-3b (Figs 6a,b and S3). 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 3·Tg-AD mice was accompanied by increased formation of p25, a cytosolic activator of cdk5, while the activation status of GSK-3b remained insignificant between the groups (Figs 6a,b and S3). The cytosolic p25 is generated through the proteolytic cleavage of p35 by calpain activity. We further determined whether brain calpain activity was increased in 9-month copper exposure in the 3·Tg-AD mice. In the copper-exposed brain, calpain activity was generally higher than control mice although it failed to show a statistical significance because of high variability among the mice (Fig. 6c). Collectively, our result indicates that coppermediated increase of phospho-tau levels closely correlated with the activation of cdk5/p25 in the brain. Copper exposure alters protein interaction with CCS Chronic copper exposure to young 3·Tg-AD mice favored amyloidogenic process of APP cleavage in the brain as measured by increasing levels of C99 and Ab. 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 CCS, an important protein that transports copper to SOD1 for its activation (Angeletti et al. 2005). To examine whether alteration of CCS binding in

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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 up-regulated in both 3- and 9-month copper exposed groups (Figs 1 and 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. With 3 months of copper exposure, the interaction of BACE1 with CCS significantly increased, whereas the interaction of SOD1 was significantly decreased (Fig. 7). On the other hand, with 9 months of 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 (Fig. S4), 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.

Discussion The effects of copper on AD pathogenesis are conflictive as some animal studies show beneficial, while others detrimental effects, (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 Ab 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



Fig. 7 Brain interaction of BACE1 or SOD1 with CCS is altered in chronic copper exposure. Steady-state levels of CCS and SOD1 were determined by immunoblotting (n = 5–6 per group). The interaction of BACE1 or SOD1 with CCS was determined by immunoprecipitating CCS followed by immunoblotting for BACE1 or SOD1. Densitometric analysis showed a significance at *p < 0.05 or **p < 0.01 compared to age-matched control group (n = 5 per group).

with a 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 3·Tg-AD mouse model, copper exposure not only increases Ab generation, particularly Ab40, but also triggers pathological tau phosphorylation and tangle formation in the brain. We demonstrate that increased Ab generation may be mediated by the up-regulation 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 c-secretase complex are concentrated, resulting in increased APP processing and the generation of Ab 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 up-regulates 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 steadystate levels of APP significantly elevated. In the 3·Tg-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 Ab 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, Ab peptides utilize them as a seed and initiate the aggregation process (Atwood et al. 1998, 2000). An in vitro study demonstrated that Ab aggregation depends on metal concentration (Hutchinson et al. 2005). Several animal studies demonstrated enhanced aggregation of Ab and plaque formation in the brain following copper or zinc exposure (Lee et al. 2002; Sparks and Schreurs 2003). Similarly, we observed elevated brain Ab, particularly Ab40 in copper-exposed 3·Tg-AD mice. At this point, it is not clear how selective generation of Ab40 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 a-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/Ab 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 a-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 3·Tg-AD mice. In addition to the direct effect of copper on APP/Ab, 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 up-regulation of BACE1 levels in copper-exposed 3·Tg-AD brain along with increasing C99 levels. Furthermore, we detected increased oxidative stress in brains with up-regulated 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.

Acknowledgements 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.).

Aß 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.

Supporting Information Additional Supporting Information may be found in the online version of this article: Figure S1 Copper exposure increases the steady-state levels of APP in SH-SY5Y cells. Figure S2 BACE1 deposition around plaques increases following chronic copper exposure. Figure S3 Densitometric analysis of steady-state levels of p35, cdk5 and GSK-3b following copper expsure in 3·Tg-AD mice. Figure S4 Chronic copper exposure increases oxidative stress in the brain. Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.

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