Trace amounts of copper in water induce β-amyloid plaques and learning deficits in a rabbit model of Alzheimer's disease

Results

Copper Induces Aβ Accumulation and Senile Plaque (SP)-Like Structures. The brains of rabbits were analyzed by using the 10D5 antibody, which shows Aβ immunoreactivity (33, 34). Cholesterol-fed rabbits with copper (0.12 ppm) added to their distilled drinking water (dH₂O/Chol/Cu) revealed a nearly 50% increase in the number of Aβ immunoreactive neurons compared with animals on a cholesterol diet and unaltered distilled water (dH₂O/Chol) (Table 1). The hippocampus, temporal cortex, and superior parietal cortex were preferentially affected compared with lateral parietal cortex, an observation that is similar to the regional distribution of SPs in AD. The superior temporal gyrus, also severely affected in AD, demonstrated a laminated distribution of Aβ immunoreactive neurons (Fig. 1B). This propensity for Aβ immunoreactive neurons to occur in layers II and IV–V is reminiscent of the laminar distribution of SPs in layers II and IV–V of the AD temporal cortex (24, 35). We also noted an increased Aβ immunoreactive staining on the meningeal surface of the brain in rabbits supplemented with copper (Fig. 1 A Right and B Right), consistent with observations in the AD brain (36). Perhaps most significantly, cholesterol-fed rabbits also showed extracellular SP-like deposits of Aβ immunoreactive material (Fig. 1 A and C). These SP-like deposits were not found among animals given distilled water (dH₂O/Chol), and rarely occur among animals on tap water (Tap/Chol, <10%; one or two per section in 1 of 16 animals). Strikingly, 75% of the cholesterol-fed animals on distilled water supplemented with 0.12 ppm copper (9 of 12) displayed SP-like deposits, and three of these rabbits had >10 SP-like deposits per histological section (Fig. 1 A).

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Fig. 1.

Trace amounts of copper induce Aβ immunoreactive neurons and SP-like structures in the brain of the cholesterol-fed rabbit. (A) Photo montage of temporal lobe and hippocampus for a cholesterol-fed rabbit given distilled water (Left) and distilled water supplemented with 0.12 ppm copper (Right) (scale bar, 1 mm). Note the numerous dark spots in the copper-supplemented rabbit; these are SP-like structures. An example is indicated by the arrowhead and is magnified in C. (B) Photo montage of superior temporal gyrus for distilled water (Left) and copper-supplemented rabbit (Right) with banding of Aβ immunoreactive neurons in layers II and IV–V (scale bar, 100 μm). (C) SP-like structure surrounded by Aβ immunoreactive neurons. Note that the plaque-like structure is somewhat removed from a blood vessel indicated by the arrowhead (scale bar, 100 μm).

Copper and Cholesterol Increase SOD and Decrease GpX. We sought to find a bloodborne marker related to elevated circulating cholesterol or altered copper levels that might co-vary with Aβ immunoreactive neurons and/or SP-like deposits in the cholesterol-fed rabbit. Free radicals and associated changes of SOD and GpX activities are known to accompany increased circulating cholesterol (37), and CP can vary with copper levels in the blood (38). Using rabbits on distilled water as the reference control (dH₂O), the activity of SOD in rabbits fed normal chow and given tap water (Tap) was marginally increased in RBCs and unchanged in the plasma (Table 2). Addition of cholesterol to the diet of animals on distilled water (dH₂O/Chol) produced a 3-fold increase in SOD activity in the plasma, and the addition of 0.12 ppm copper (dH₂O/Chol/Cu) produced an almost 4-fold increase in SOD activity (Table 2). Among the cholesterol-fed rabbits given copper sulfate in their distilled water, the activity of GpX decreased significantly compared with cholesterol-fed animals given distilled water. Although there was a suggested increase in CP, there was no significant effect of circulating cholesterol levels, concentration of copper in the drinking water, and levels of a CP in rabbit blood.

Copper-Induced Aβ Retards Trace Conditioning but Not Delay Conditioning. To assess the effects of copper-induced Aβ accumulation on learning, memory, and sensory processing, we devised and administered a battery of behavioral tests to a subset of the cholesterol-fed rabbits. The tests included a measure of sensitivity and responsiveness to air puff, a difficult learning task, an easy learning task, and a task designed to measure their hearing. We chose trace classical conditioning of the rabbit nictitating membrane response as the difficult learning task because it engages the hippocampus and temporal lobe (39, 40), areas in which we noted the highest levels of Aβ accumulation. Measures of the average percent CR to the tone across 8 days of hippocampally dependent tone–air puff trace conditioning showed that 0.12 ppm copper had a profound effect on the ability of cholesterol-fed rabbits to acquire CR (Fig. 2A). In fact, rabbits with copper in their drinking water (dH₂O/Chol/Cu) did not exceed an average level of 15% CR, whereas rabbits on distilled water (dH₂O/Chol) acquired CR approaching average levels of 60%. In strong contrast, when rabbits given copper were shifted to a simpler, hippocampally independent, short-delay conditioning paradigm, they were able to acquire CR to levels approaching 80% (Fig. 2B). Equally important, all rabbits showed comparable levels of sensitivity and responsiveness to the air puff and to the tone. Specifically, there were no significant differences between the copper-supplemented and distilled water groups in any of the dependent variables used to assess sensitivity and responsiveness to air puff, including response frequency, amplitude, latency, or area (P values >0.07). An analysis of hearing measured as percent CR to seven different tone intensities (60–90 dB) indicated that there were no between-group differences in the levels of responding to the tone intensities (P values >0.49) (Fig. 2C).

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Fig. 2.

Effects of trace amounts of copper (0.12 ppm) induce deficits in trace but do not delay classical conditioning of the rabbit nictitating membrane response. (A) Trace conditioning. Mean (± SEM) percent CR for cholesterol-fed rabbits with distilled water (dH₂O/Chol) and with copper sulfate (dH₂O/Chol/Cu) added to their distilled water revealed a significant overall copper-induced learning deficit as a main effect of groups [F (1,9) = 7.37, P < 0.05] and a highly significant groups–sessions interaction [F (7,63) = 4.81, P < 0.001]. (B) Delay conditioning of the rabbit nictitating membrane response showed that there was no difference in rate or level of learning of a simpler task between rabbits given copper in their distilled water or distilled water alone. (C) Tone intensity testing after delay conditioning showed no differences in the ability of rabbits given copper in their distilled water or distilled water alone to hear the tone used during trace conditioning.

Discussion

Copper and AD. The present data provide strong support for the suggestion that copper is implicated in the accumulation of Aβ (8, 10). A recent report described APP as an exporting system for copper and suggested that elevated levels of APP in the brain may remove increased copper (11). Indeed, as we have already noted, chelation of copper that would prevent Aβ accumulation has been suggested as a potential therapy for AD (9, 13–15). Clioquinol, a copper–zinc chelator, is in phase I (13) and phase II clinical trials (16). In contrast to Aβ's potential neuronal role in clearing copper, copper may influence clearance of Aβ from the brain at the level of the interface between the blood and cerebrovasculature.

The Environmental Protections Agency maximum contaminant level goal (MCLG) for copper in drinking water is set at 1.3 ppm (1.3 mg/liter) copper or corrosives liberating 1.3 ppm copper, and treatment plants that exceed this MCLG are required to monitor the water and implement techniques that will reduce its corrosiveness (30). The MCLG is based on the lowest observed adverse health effect level (LOAEL) of 5.3 mg/day, a level which may induce gastrointestinal distress (30). Interestingly, although the dietary reference intake [DRI, formerly called recommended daily allowance (RDA)] for copper in adults is only 0.9 mg/day, the normal tolerable upper limit is considered to be 10 mg/day (7). Cholesterol-fed rabbits in the present experiment were allowed 0.12 ppm or 0.12 mg/liter, and given that rabbits drink ≈300–600 ml of water per day, they consumed between 0.04 and 0.08 mg of copper per day. The present data show that this level of copper consumption was sufficient to induce significant Aβ accumulation and the formation of significant SP-like structures in the cholesterol-fed rabbit. Although we can only speculate about how the effects of copper consumption in cholesterol-fed rabbits relate to those in humans, it is of note that the levels of copper in the cholesterol-fed rabbit drinking water that induced Aβ and SP-like structures are well below those considered safe for humans.

A Cholesterol-Fed Rabbit Model of AD. Unlike transgenic mouse models, which have failed to produce more than one or two signs of AD pathology (41), the cholesterol-fed rabbit shows pathologies consistent with at least 12 different features of the AD brain. These include the currently described neuronal accumulation of Aβ immunoreactivity (17, 19), extracellular Aβ plaques (23), and meningeal Aβ immunoreactivity (36), as well as previously noted apolipoprotein E immunoreactivity (18, 22), cathepsin D immunoreactivity (20, 23), SOD immunoreactivity (23), microgliosis (21, 23), apoptosis (21), vascular activation of SOD (24), mouse endothelial cell antigen (MECA-32) immunoreactivity (25), breaches of the blood brain barrier (25), elevated brain cholesterol (23), and elevated Aβ concentration (24). To these indices of pathology we can now add a deficit in the ability to learn a difficult task that depends on the temporal cortex and hippocampus.

Conclusions

The addition of trace amounts of copper to the drinking water of cholesterol-fed rabbits induces accumulation of Aβ, formation of SP-like structures, reduction of GpX activity, increases in SOD activity, and retardation of the rabbit's ability to learn a difficult task. Thus, there may be a relationship between the diminished ability to inactivate peroxide due to reduced GpX activity in the blood and increased neuronal accumulation of Aβ immunoreactivity, formation of SP-like structures in the neuropil, and observed deficits in complex memory acquisition. Overall, we would suggest that cholesterol entering the brain from the circulation of cholesterol-fed rabbits induces the neuronal accumulation of Aβ, and that copper influences clearance of Aβ from the brain at the level of the interface between the blood and cerebrovasculature.