Zinc Overload Enhances APP Cleavage and Aβ Deposition in the Alzheimer Mouse Brain

Background Abnormal zinc homeostasis is involved in β-amyloid (Aβ) plaque formation and, therefore, the zinc load is a contributing factor in Alzheimer's disease (AD). However, the involvement of zinc in amyloid precursor protein (APP) processing and Aβ deposition has not been well established in AD animal models in vivo. Methodology/Principal Findings In the present study, APP and presenilin 1 (PS1) double transgenic mice were treated with a high dose of zinc (20 mg/ml ZnSO4 in drinking water). This zinc treatment increased APP expression, enhanced amyloidogenic APP cleavage and Aβ deposition, and impaired spatial learning and memory in the transgenic mice. We further examined the effects of zinc overload on APP processing in SHSY-5Y cells overexpressing human APPsw. The zinc enhancement of APP expression and cleavage was further confirmed in vitro. Conclusions/Significance The present data indicate that excess zinc exposure could be a risk factor for AD pathological processes, and alteration of zinc homeostasis is a potential strategy for the prevention and treatment of AD.


Introduction
The presence of extracellular b-amyloid (Ab) plaques in the brain is one of the pathological hallmarks of Alzheimer's disease (AD). Mounting evidence has demonstrated that aberrant zinc homeostasis is involved in the pathogenesis of AD [1,2,3,4]. In the post-mortem AD brain, a marked accumulation of zinc is found in the Ab plaques [5,6,7,8,9,10]. Since Ab peptide has zinc-binding sites, and zinc is the only physiologically available metal able to precipitate Ab, the abnormal enrichment of zinc in the AD brain indicates that zinc binding to Ab plays a role in the formation of amyloid plaques [11]. Furthermore, zinc chelating agents, such as clioquinol (CQ) and DP-109, that modulate brain zinc levels can inhibit the formation of amyloid plaques [12,13,14]. In preliminary studies, CQ has shown some effects on cognition in AD patients [15,16,17]. Thus, abnormal zinc homeostasis is believed to be a contributing factor leading to Ab aggregation, and alteration of zinc homeostasis is a potential therapeutic strategy for AD.
The disruption of zinc homeostasis in the AD brain is associated with the aberrant distribution and altered expression of zincregulating metalloproteins, such as metallothionein, zinc transporters (ZnT) and divalent metal transporter 1 (DMT1). We have reported that high levels of ZnT1, 3-7 and DMT1 proteins are located in the degenerating neurites in or around the Ab-positive plaques associated with human AD and the APP/presenilin 1 (PS1) transgenic mouse brain [18,19,20,21]. Significant alterations in the expression levels of ZnT1, 4, and 6 have been detected in AD postmortem brain specimens [22,23]. Genetic abolition of ZnT3 results in disappearance of zinc ions in the synaptic vesicles [24], and leads to an age-dependent deficit in learning and memory in ZnT3 knockout mice [25]. Most interestingly, a markedly reduced plaque load and less insoluble Ab have been observed in ZnT3 knockout plus APP overexpressed mouse brain [26], suggesting a role of synaptic zinc in Ab generation and aggregation. Furthermore, in vitro studies have shown that both APP and its proteolytic product Ab contain zinc binding domains. However, the involvement of zinc in APP processing and Ab deposition has not been well established in AD transgenic models in vivo.
In the present study, we extended our experiments to examine whether chronic intake of water containing a high level of zinc accelerates Ab deposition and APP cleavage in APP/PS1 mouse brain. We found that a high level of dietary zinc could cause cognition dysfunction and enhance the aggregation of Ab. Furthermore, we found that a high level of zinc also enhanced Ab generation through altering the expression levels of APP and APP cleavage enzymes in vivo and in vitro. Our data support the possibility that dietary zinc overload has the potential to be a contributing factor to the pathophysiology of AD.

Results
Chronic high intake of dietary zinc induces spatial learning-memory deficits in APP/PS1 mice APP/PS1 transgenic mice at the age of 3 months were given a standard diet and deionized water containing ZnSO 4 (20 mg/ml). Morris water maze tests were performed to evaluate whether high dietary zinc treatment affects learning and memory in APP/PS1 mice at the age of 9 months. These included 2 days of visible platform training, 5 days of hidden platform tests, and a probe trial 1 day after the last hidden platform test ( Figure 1). The visible platform tests showed that the zinc group and control mice had a similar escape latency and path length (p.0.05; Figure 1A, B), suggesting that zinc treatment did not significantly affect motility or vision in the transgenic mice. In the place navigation (hidden platform) tests, the zinc group mice showed a longer escape latency and a longer path length before swimming onto the hidden platform compared with the control mice fed a normal diet (p,0.01; Figure 1A, B). Furthermore, the probe trial showed that the number of times the mice traveled into the center of the northwest quadrant, where the hidden platform was previously placed, was significantly less for zinc group mice compared with controls (p,0.01; Figure 1C). Taken together, these data suggest that high-dose oral zinc leads to spatial learning-memory impairments in APP/PS1 mice.
Enhancement of zinc content in serum and brain of APP/ PS1 mice fed a zinc diet The serum zinc levels were measured in the transgenic mice at the age of 9 months. There was a significant increase in zinc level in the zinc group (11.2162.42 mg/ml), compared with the control (0.4360.12 mg/ml) (p,0.01; Figure 2A). We also measured the zinc level in the brain of the transgenic mice to determine the effects of a high zinc diet. The brain zinc level was 173.17624.72 ng/mg in the zinc group, and 23.5962.31 ng/ mg in the control group. Statistical analysis showed that treatment with a high dose of zinc significantly increased the level of zinc in the brain (p,0.01; Figure 2B).
Increase in the number of zinc-containing plaques in the brain of APP/PS1 mice fed a high zinc diet The autometallography (AMG) procedure allows demonstration of a striking condensation of ionic zinc within plaques in the human postmortem AD brain and the APP/PS1 transgenic mouse brain [10,19]. Brain sections of APP/PS1 mice given a high dose of zinc in their drinking water and normal diet were subjected to AMG analysis. In general, zinc-positive plaques were distributed throughout the cortex and hippocampus in all examined animals ( Figure 3A), as previously described [27]. Both the number and size of the zinc-positive plaques in the zinc-treated group were markedly increased in the cortex and hippocampus ( Figure 3A). Statistical analyses showed that high zinc treatment significantly increased the number of zinc-positive plaques by 146.24612.30% in the cortex and 225.00622.97% in the hippocampus respectively, compared with the control group (p,0.01; Figure 3B). The size of zinc-containing plaques in the brain of APP/PS1 mice fed a high zinc diet was increased by 186.83615.74% Figure 1. Morris water maze assessment of APP/PS1 transgenic mice. APP/PS1 mice at the age of 3 months were given either a standard diet and deionized water (Con), or a standard diet and deionized water containing 20 mg/ml ZnSO4 (Zn). Morris water maze tests were performed to evaluate whether high dietary zinc treatment affects learning and memory in APP/PS1 mice at the age of 9 months. (A, B) In the visible platform training from day 1 to 2, mice in different groups exhibited a similar escape latency and path length to find the visible platform. At day 3, 5, and 7 of the hidden platform tests, Zn-treated mice showed the longest latency and escape length. (C) In the probe trial on the last day, the Zn-treated mice exhibited the lowest passing times into the northwest quadrant, where the hidden platform was previously located. * p,0.05, ** p,0.01 versus control group (repeated measures ANOVA). doi:10.1371/journal.pone.0015349.g001 in the cortex and 179.60621.74% in the hippocampus, compared with the control group (p,0.01; Figure 3B).

High dietary zinc increases Ab burden in the APP/PS1 mouse brain
To determine whether chronic high intake of dietary zinc potentiates Ab deposition, brain sections of APP/PS1 mice were subjected to Ab immunohistochemical analysis. As shown in Figure 4, both the number and size of the Ab-immonoreactive senile plaques were markedly increased in the cortex and hippocampus in the brain of zinc-treated mice ( Figure 4A). Statistical analyses showed that a high intake of dietary zinc significantly increased the number of Ab plaques by 154.5568.25% in the cortex and 188.31611.90% in the hippocampus, compared with the control group fed a normal diet (p,0.01; Figure 4B). We also evaluated the changes in Ab burden by measuring the areas of Ab-positive neuritic plaques in the mouse brain. In the zinc group, the area of Ab plaques was significantly increased by 173.36611.44% in the cortex and 213.15634.29% in the hippocampus, compared with the control group (p,0.01; Figure 4B).
Increase in expression level of APP protein and Ab generation in the APP/PS1 mouse brain after a high zinc diet To further test whether a high dose of dietary zinc affects APP expression, the levels of APP mRNA and APP695 protein were measured by RT-PCR and Western blotting, respectively. As can be seen in Figure 5A, the expression levels of APP mRNA were not significantly changed between groups after examining brain samples of APP/PS1 mice treated with zinc or fed a normal diet. Western blot analysis revealed that zinc treatment significantly increased the level of APP695 protein by 132.79616.82%, compared with controls fed a normal diet ( Figure 5B).
To determine whether a high zinc intake had altered brain Ab levels in APP/PS1 mice, a Sandwich ELISA for the detection of Ab was employed ( Figure 5C, D). Statistical analysis showed that a high zinc diet significantly increased the level of Ab1-40 (p,0.05) and Ab1-42 (p,0.01) in the brain, compared with controls. The levels of Ab1-40 and Ab1-42 were increased by 122.19620.60% ( Figure 5C) and by 148.96615.67% ( Figure 5D) in the brain homogenates of APP/PS1 mice with a high level of zinc in their drinking water, compared with controls fed a normal diet.
High level of zinc in drinking water accelerates APP processing in the APP/PS1 mouse brain To further examine whether a high intake of dietary zinc altered APP processing, the relative key enzymes (including ADAM10, BACE1 and PS1) and cleavage fragments of APP (including sAPPa, sAPPb, C83 and C99) in the brain samples of APP/PS1 mice were subjected to Western blot analyses. As shown in Figure 6A, zinc treatment significantly decreased the expression level of ADAM10 by 45.8067.01% compared with the control group (p,0.01; Figure 6A). In contrast, the level of BACE1 was significantly increased in zinc-treated mouse brain by 147.49621.91% (p,0.01; Figure 6A). Furthermore, the level of PS1 in zinc-treated mouse brain was significantly increased by 130.44636.80%, compared with the control group (p,0.01; Figure 6A).
We then examined the levels of a-secretase-generated sAPPa/ C83 and b-secretase-generated sAPPb/C99 fragments in the transgenic mouse brain. Zinc treatment significantly reduced the level of sAPPa by 53.5563.32% (p,0.01; Figure 6B), and increased the level of sAPPb by 255.62627.24%, compared with the control group (p,0.01; Figure 6B). The level of C83 fragments was reduced by 70.4864.27% and the level of C99 was increased by 144.65615.79% in brain of zinc-treated mice relative to the controls (p,0.01; Figure 6B). Taken together, these results indicate that the a-secretase cleavage activity is markedly reduced while band c-secretase are increased in the brain of zinc-treated transgenic mice.

High zinc exposure enhances amyloidosis in APPsw transfected cells
To further verify that high zinc exposure might be involved in APP processing and Ab secretion, a human neuroblastoma SHSY-5Y cell line stably transfected with APPsw was used as an in vitro model [28,29]. A zinc concentration of 1 mM (low zinc) and 70 mM (high zinc), and a concentration of TPEN (zinc chelator) of 1 mM were selected based on the evaluation of cell viability by MTT assay (Figure 7A, B). The zinc-specific fluorescent probe, Zinquin, was used to examine the levels of zinc in cells after zinc or chelator treatments. The results showed that Zinquin fluorescence was distributed in a punctate pattern in APPsw cells ( Figure 7C). Zinc treatments increased the Zinquin fluorescence, while TPEN reduced it ( Figure 7C). The fluorescence density assays showed that 70 mM zinc treatment significantly enhanced the Zinquin fluorescence by 300.31656.19% (p,0.01; Figure 7D), whereas TPEN treatment reduced the fluorescence density by 30.94614.65% compared with the control cultures (p,0.01; Figure 7D). Consistent with our in vivo data from APP/PS1 transgenic mice, high zinc (70 mM) exposure significantly increased the APP cleavage enzyme levels of BACE1 by 135.50617.05% and PS1 by 132.79613.11% (p,0.01), and reduced the levels of ADAM 10 by 80.1165.04% (p,0.05), respectively, in APPsw cells ( Figure 8A). Subsequently, the levels of b-secretase-generated fragments sAPPb and C99 were markedly increased by 131.4668.57% and 126.95622.46% (p,0.01), while the levels of a-secretase-generated sAPPa and C83 were decreased by 79.8964.63% (p,0.01) and 76.9465.61% (p,0.05), respectively, following 70 mM zinc treatment ( Figure 8B). Also, chelation of zinc with 1 mM TPEN reversed the changes in the expression levels of the cleavage enzymes and fragments of APP ( Figure 8B). Furthermore, ELISA detection showed that the levels of secreted Ab1-42 in culture medium were increased by 166.27623.04% in the 70 mM zinc treatment group (p,0.01; Figure 8C), and reduced by 37.18610.36% with 1 mM TPEN (p,0.01; Figure 8C), compared with the control group. These data clearly indicated that high zinc exposure enhanced the amyloidogenic APP cleavage pathway and Ab generation in APPsw overexpressing cells.
In contrast to high zinc exposure, low zinc (1 mM) treatment guided APP processing to the non-amyloidogenic pathway in our APPsw overexpressing cells. Following 1 mM zinc treatment, the levels of AMAM10, sAPPa and C83 were increased by 142.21612.04% (p,0.05; Figure 8A), 116.71619.07% (p,0.05; Figure 8B) and 117.81612.47% (p,0.05; Figure 8B), whereas the levels of BACE1, sAPPb and C99 were reduced by 89.61613.34% (p,0.05; Figure 8A), 81.0364.80% (p,0.05; Figure 8B) and 94.0669.89% (p,0.05; Figure 8B), respectively, compared with the controls. The level of secreted Ab1-42 in culture medium was decreased by 64.43611.77%, following 1 mM zinc treatment (p,0.05; Figure 8C). These data are consistent with previous reports showing that exposure to low concentrations of zinc (#50 mM) significantly enhances the levels of secreted APP and results in reduced release of Ab in a medium of cultured CHO-K1 cells [30,31].

Discussion
Both APP and its proteolytic byproduct Ab, which play central roles in senile plaque formation in the pathogenesis of AD, are zinc-containing metalloproteins that contain zinc-binding domains [32]. Therefore, it is rational to speculate that zinc overload may be involved in APP expression, Ab generation and aggregation. In the present study, involving treatment with a high level of zinc in the drinking water of APP/PS1 mice, we found that mice fed a high zinc diet exhibited spatial learning impairments as shown by Morris water maze tests. Apart for body weight loss, fur color changes (data not shown), raised serum and brain zinc levels, and a high zinc content in the drinking water resulted in no other overt signs of toxicity such as general behavioral and neurological changes during the entire observation period which our model mice were given a high zinc diet. This is in agreement with previous reports showing that there was no serious toxicity in C57BL/6 mice after chronic zinc treatment at the same dose [33,34]. Thus, we further evaluated the effects of a chronic high dietary zinc intake on accumulation of Ab deposits, as well as APP expression and cleavage in the APP/PS1 transgenic mouse brain.
We and others have reported that zinc is highly concentrated in amyloid plaques in human postmortem brain samples [5,6,7,8,10,18,35] and in AD transgenic mouse brains [9,36]. Here, we Figure 8. Expression level of APP cleavage enzymes and products in APPsw cells. SHSY-5Y cells stably overexpressing APPsw were exposed to 1 mM zinc, 70 mM zinc, 1 mM TPEN, and 70 mM zinc plus 1 mM TPEN, respectively, for 8 h. (A) Western blot was performed to determine the expression levels of APP cleavage enzymes, including ADAM10, BACE1 and PS1. GAPDH was used as an internal control. Low (1 mM) and high zinc (70 mM) treatment showed different effects on the expression of ADAM10. ADAM10 was markedly increased after low zinc treatment, but significantly reduced after high zinc treatment. There were no significant changes in ADMA10 levels in the TPEN or Zn + TPEN group, compared with controls. High zinc (70 mM) treatment significantly increased the levels of BACE1 as well as PS1. TPEN treatment reduced the BACE1 and PS1 levels. In Zn + TPEN group, the levels of BACE1 and PS1 were significantly reduced compared with the high zinc (70 mM) treatment group. (B) The expression levels of APP cleavage products, including sAPPa, sAPPb, C83 and C99, were determined by Western blot analysis. GAPDH was used as an internal control. Low zinc (1 mM) exposure significantly enhanced the expression level of sAPPa, however, high zinc (70 mM) treatment markedly reduced the sAPPa expression level. There were no significant changes in sAPPa levels in the TPEN or Zn + TPEN treatment group compared with controls. The expression level of sAPPb was significantly decreased after low zinc (1 mM) treatment, but was significantly increased after high zinc (70 mM) found that a high intake of dietary zinc resulted in an increase in zinc-containing plaques in APP/PS1 transgenic mice. Coincident with the AMG results, Ab immunohistochemical analyses demonstrated that there was an increased Ab burden in transgenic mice fed a high zinc diet. Since the small peptide Ab possesses selective high-and low-affinity zinc binding sites [32,37], and zinc at a concentration of 300 nM can rapidly destabilize Ab and result in fibril formation [11,37], it is likely that an overload of brain zinc increases Ab binding and, hence, enhances Ab aggregation and plaque formation in the brain after chronic administration of a high zinc diet.
Zinc is toxic and, besides its physiological roles, it is involved in neuronal and glial death through activation of multiple intracellular pathways leading to necrotic, apoptotic and autophagic neuronal death [38,39,40,41]. The elevated level of zinc in the AD brain is caused, at least partly, by the abnormal distribution and expression of zinc-regulating proteins such as ZnTs and DMT1 [18,19,21]. At an early stage of AD, the elevated brain zinc results in the formation of zinc-Ab complex, which is of some benefit in protecting against zinc toxicity [42,43,44]. On the other hand, recent studies have shown that soluble Ab is a major factor in neuronal and synaptic pathology, since it is more toxic than insoluble Ab [45,46,47]. It is likely that the initial zinc-Ab complex and subsequent Ab aggregation inhibits Ab mediated neurotoxicity. However, it is worth noting that the initial zinc-Ab complex may serve as a seed for the process of Ab aggregation and plaque formation in the brain [5,11,48,49]. Although it is still debatable whether Ab aggregation mediated by interaction with zinc plays a role in reducing the toxicity of soluble Ab or whether the zinc-containing plaques themselves are toxic to neuronal cells [44,50,51,52], the interaction between Ab and zinc seems to be a critical factor for activating AD pathological processes. Nevertheless, our present data suggest that a high zinc intake leads to more zinc-Ab complex formation, accelerates Ab deposition and enhances the amyloid burden. Further studies are needed to elucidate the paradoxical role of zinc in plaque pathology [31].
APP protein contains a novel zinc binding motif which is located between the cysteine-rich and negatively charged ectodomains [32]. Besides its structural role, zinc may be involved in the function and metabolism of APP protein, and produce an even greater deposition of Ab. However, apart for several in vitro studies that tested the effects of zinc on APP processing [30,53], there are no detailed reports whether zinc binding to APP alters APP processing and Ab production in AD transgenic animal models. In the present study, we found that a high intake of dietary zinc significantly increases the expression levels of APP protein in APP/ PS1 transgenic mouse brain. We also found that high-dose zinc treatment results in reduced expression levels of ADAM10, but enhances the levels of BACE1 and PS1, resulting in increased secretion of sAPPb over sAPPa in the transgenic mouse brain. Further, consistent with our in vivo data, high zinc (70 mM) exposure suppresses a-secretase cleavage, but enhances band csecretase cleavage of APP and Ab generation in APPsw overexpressing cells. Thus, our in vivo and in vitro studies clearly show that high-dose zinc treatment enhances the amyloidogenic APP cleavage pathway and Ab secretion. Interestingly, a recent study involving APP/PS1 mice fed a zinc-deficient diet has shown that such a diet increases the plaque volume but does not alter the total plaque number in the brain [27]. Chronic high zinc-or copper-treated mice overexpress APP-C100, which contains Ab but not the N-terminal zinc and copper binding domain of APP, resulting in reduced soluble Ab levels but with no changes in the total Ab levels in the brain [54]. It has also been reported that exposure to copper and, presumably, a mixture of other metals in drinking water results in enhanced Ab deposition in the brains of rabbits fed a high cholesterol diet [55]. Alain Boom and colleagues showed that 100 mM zinc induced the appearance known to be associated with increased tau phosphorylation, suggesting that zinc plays a considerable role in the development of tau pathology associated to Alzheimer's disease [56]. On the other hand, some reports have shown the protective effects of low micromolar concentrations of zinc against Ab cytotoxicity [43,44,57]. So far, the role of zinc in AD remains debatable. Both high and low zinc could play a harmful role. Whether or not to supply zinc and what the suitable dose range should be are topics worthy of future research on AD. Taking these findings together with the present evidence that high-dose zinc treatment leads to enhanced amyloidogenic APP cleavage and Ab aggregation in the APP/ PS1 mouse brain and APPsw overexpressing cells, it can be concluded that disturbed metal homeostasis is involved in multiple steps of APP processing and Ab deposition by a series of complicated mechanisms.
In summary, the present study provides evidence that chronic exposure to high zinc levels in drinking water leads to an increase in APP expression, amyloidogenic APP cleavage and Ab deposition in the APP/PS1 transgenic mouse brain. The present data, together with previous reports, suggest that excess zinc exposure could be a risk factor for AD pathological processes, and corrections of metal abnormalities in the brain are beneficial strategies for AD prevention and therapy.

Ethics statement
The experimental procedures were carried out in accordance with the regulations of the animal protection laws of China and approved by the animal ethics committee of China Medical University (JYT-20060948). All efforts were made to minimize animal suffering and the number of animals used.

Animals and Treatments
Male APP/PS1 double transgenic mice (B6C3-Tg (APPswe, PSEN1dE9) 85Dbo/J mice) were used in the present study (breeding pairs were obtained from the Jackson Laboratory, West Grove, PA). They were kept in cages in a controlled environment (22-25uC, 50% humidity). Mice at the age of 3 months were randomly assigned to one of two groups (n = 12 in each group). (1) Control group: mice were given a standard diet (30 ppm zinc) and deionized water ad libitum. (2) Zinc group: mice were given a standard diet and deionized water containing ZnSO 4 (20 mg/ml). The doses of zinc chosen for this study were based on previous reports showing that it produced no serious toxicity in C57BL/6 mice after chronic treatment [33]. The potential toxicity of highdose oral zinc on transgenic mice was evaluated by monitoring their general aspect and body weight. As reported previously [33,34], apart for a coat color change from black to bright brown on the back, and a decline in body weight starting from the 14th week after zinc treatment, no other overt signs of toxicity, such as general behavioral and neurological changes, were observed in mice on a high zinc diet during the entire observational period. At the age of 9 months, blood samples were drawn from the heart just prior to decapitation, and the levels of zinc in serum and brain were analyzed using a polarized Zeeman atomic absorption spectrophotometer (Hitachi 180-80, Japan).

Morris Water Maze
Morris water maze tests were carried out as previously described with few modifications [58], using a circular tank, equipped with a digital pick-up camera to monitor the animal behavior and a computer program for data analysis (ZH0065, Zhenghua Bioequipments, China). Briefly, one week before reaching the age of 9 months, transgenic mice were trained for 2 days to remember the visible platform, which is placed in the center of the northwest quadrant in the tank with opaque water. From the 3rd to 7th day, the platform was placed just below the water surface (hidden platform) for the place navigation test, and each mouse was subjected to 3 trials per day with an inter-trial interval of 1 min. For each trial, the latency to escape to the hidden platform and the path length were recorded. At the 8th day, the platform was removed from the tank for the probe trial. The number of times the animal crossed the center of the northwest quadrant at an interval of 1 min was recorded. Finally, data for the escape latency, the path length and the number of passing times between groups were analyzed statistically.

Tissue Preparation
The day after the Morris water maze tests, mice were anaesthetized with sodium pentobarbital (50 mg/kg, i.p.) and sacrificed by decapitation. The brains were removed immediately and split sagittally into halves. The left hemisphere was kept at 280uC for Western blotting, RT-PCR or ELISA analyses. The right hemisphere was further cut into two slabs. One was immersed in 3% glutaraldehyde for zinc autometallographic analysis while the other was placed in 4% paraformaldehyde for immunohistochemical staining.

Autometallography and Stereological Assessment
AMG was performed to analyze the distribution of zinc in the transgenic mouse brain according to our previous reports [20,59]. In brief, brain slices (2 mm) were cut with a vibratome and immersed in a mixture of 3% glutaraldehyde and 0.1% sodium sulfide at 4uC for 2 days. The slices were placed in 30% sucrose, frozen with liquid nitrogen and 30-mm cryostat sections were prepared. Brain sections containing typical hippocampal structures from 2 groups were selected, placed in the same jar, and incubated in AMG developer at 26uC for 60 min. The AMG development was stopped by rinsing with 5% sodium thiosulfate for 10 min. After several washes with distilled water, the sections were dehydrated, covered with neutral balsam and examined with a light microscope equipped with a digital color camera. The sodium diethyldithiocarbamate trihydrate (DEDTC, Merck, 6689) control procedures were performed to ensure the specificity of the zinc ion staining [59].
The stereological assessment of zinc load in the AMG-stained brain sections was performed as reported previously [18], and this was carried out in matched brain areas from different groups. Briefly, a set of every 6th systematically sampled 30-mm-thick AMG-stained sections of the transgenic mouse brain (yielding typically 5 sections/mouse) was selected. The total number of zincpositive plaques was counted using the optical fractionator technique, while the area of zinc-positive plaques was analyzed in the same sections using the area fraction technique. The data were analyzed with Image-Pro Plus 6.0 software (Media Cybernetics, USA).

Immunohistochemistry and Ab Load Measurements
Routine free floating ABC procedures were applied. Cryostat sections (30 mm) were treated with 3% hydrogen peroxide (H 2 O 2 ) in PB for 10 min to reduce endogenous peroxidase activity. After rinsing, the sections were treated with 5% bovine serum albumin and 3% goat serum in TBS for 1 h. They were then incubated with mouse anti-Ab (1:500, Sigma, A5213) overnight at 4uC. After rinsing in TBS, the sections were incubated with 1:200 diluted biotinylated goat anti-mouse IgG for 1 h at room temperature (RT). The sections were then rinsed and treated with an ABC kit for 1 h at RT. The sections were rinsed in TBS and incubated with 0.025% 3, 3-diaminobenzidine (DAB) plus 0.0033% H 2 O 2 in TBS for 10 min. After rinsing, the sections were dehydrated, covered with neutral balsam, and examined with a light microscope. Control sections were incubated with normal serum instead of Ab antibody followed by all subsequent incubations as described above. The stereological assessment of Ab load in the Ab-immunostained brain sections was performed according to the above described procedure.

Cell Culture and Drug Treatment
Human neuroblastoma SHSY-5Y cells stably transfected with human APPsw [28,29] were grown in DMEM supplemented with 10% heat-inactivated fetal calf serum, 100 U/ml penicillin, 100 mg/ml streptomycin and 200 mg/ml G418 at 37uC in humidified 5% CO 2 air. Cells were incubated in serum-free medium for 2 h when they reached nearly 70% confluence. Then, cells were incubated with ZnSO 4 (1 mM, 70 mM), TPEN (1 mM) or ZnSO 4 (70 mM) plus TPEN (1 mM) for 8 h. The concentrations of zinc and TPEN were selected based on routine MTT assay. To analyze the zinc entry into the cells, the staining with a zincspecific fluorescent dye, Zinquin, was carried out by incubating the cells with 0.24 mM Zinquin ethyl ester (Alexis, USA) for 30 min, and then examining them under a fluorescent microscope equipped with an image analysis system.

RT-PCR
Total RNA was isolated using Trizol reagent (Invitrogen) after homogenizing the brain tissue samples. The isolated RNA was examined by UV-spectroscopy at 260 nm. Total RNA (2 mg) of each sample was first transcribed to cDNA using a Reverse Transcription System kit (Promega, Madison, WI, USA). PCR amplification was performed with reagents from Promega. The cDNA solution was amplified with primers based on the human APP sequences. The primer sequences were: APP: 59-GACT-GACCACTCGACCAGGTTCTG-39 (upstream), 59-CTTGAA-GTTGGATTCTCATACCG-39 (downstream); GAPDH: 59-A-CGGATTTGGTCGTATTGGG-39 (upstream), 59-CGCTCCT-GGAAGATGGTGAT-39 (downstream). Amplification was performed as follows: APP: 35 cycles of 95uC for 30 s, 62uC for 30 s and 72uC for 30 s; GAPDH: 30 cycles of 95uC for 45 s, 58uC for 45 s, and 72uC for 60 s. The PCR products were normalized in relation to standards of GAPDH mRNA. The results were determined and quantified with ChemDoc XRS Quantity One software.

ELISA
Ab levels were determined using ELISA assay as described previously [21]. In brief, samples were placed in a 1:8 dilution of 5 M guanidine HCl/50 mM Tis HCl and thoroughly ground. The homogenate was diluted with dilution buffer containing an inhibitor protease complex and centrifuge at 12,000 rpm for 30 min at 4uC. The samples were then loaded on to 96-well plates and the level of Ab was determined using ELISA kits for Ab1-40 (Kuregen, KU0821E-10) and Ab1-42 (Invitrogen, KHB 3441), according to the manufacturers' protocols. The absorbance was recorded at 450 nm using a 96-well plate reader.

Statistical Analysis
Data are expressed as means 6 SEM. For water maze analysis of latencies and path length, repeated measures analysis of variance (ANOVA) were performed, and differences among means were evaluated with multivariable ANOVA. Other comparisons were analyzed by Student's t test, and one-way ANOVA Post hoc Fisher's PLSD for cultured cells. All data were analyzed using SPSS 13.0 software, and statistical significance was assumed if p,0.05.