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J. Biol. Chem., Vol. 275, Issue 26, 19439-19442, June 30, 2000

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ACCELERATED PUBLICATION
Evidence that the -Amyloid Plaques of Alzheimer's Disease Represent the Redox-silencing and Entombment of A by Zinc^*

Math P. Cuajungco^§¶, Lee E. Goldstein, Akihiko Nunomura^**, Mark A. Smith, James T. Lim, Craig S. Atwood, Xudong Huang, Yasser W. Farrag, George Perry, and Ashley I. Bush

From the  Laboratory for Oxidation Biology, Genetics and Aging Unit, Massachusetts General Hospital, Charlestown, Massachusetts 02129, the ^§ Department of Psychiatry and Behavioral Science, University of Auckland School of Medicine, Auckland, New Zealand, the  Department of Pathology, Case Western Reserve University, Cleveland, Ohio 44106, and the ^** Department of Psychiatry and Neurology, Asahikawa Medical College, Asahikawa 078-8510, Japan

Received for publication, March 15, 2000, and in revised form, April 27, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A binds Zn²⁺, Cu²⁺, and Fe³⁺ in vitro, and these metals are markedly elevated in the neocortex and especially enriched in amyloid plaque deposits of individuals with Alzheimer's disease (AD). Zn²⁺ precipitates A in vitro, and Cu²⁺ interaction with A promotes its neurotoxicity, correlating with metal reduction and the cell-free generation of H₂O₂ (A1-42 > A1-40 > ratA1-40). Because Zn²⁺ is redox-inert, we studied the possibility that it may play an inhibitory role in H₂O₂-mediated A toxicity. In competition to the cytotoxic potentiation caused by coincubation with Cu²⁺, Zn²⁺ rescued primary cortical and human embryonic kidney 293 cells that were exposed to A1-42, correlating with the effect of Zn²⁺ in suppressing Cu²⁺-dependent H₂O₂ formation from A1-42. Since plaques contain exceptionally high concentrations of Zn²⁺, we examined the relationship between oxidation (8-OH guanosine) levels in AD-affected tissue and histological amyloid burden and found a significant negative correlation. These data suggest a protective role for Zn²⁺ in AD, where plaques form as the result of a more robust Zn²⁺ antioxidant response to the underlying oxidative attack.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Amyloid- protein (A; 39-43 amino acid residues, 4 kDa) is the main constituent of neuritic and diffuse plaques and also cerebrovascular amyloid deposits that characterize the neuropathology of Alzheimer's disease (AD)¹ (1, 2). Both A deposition in the neocortex, and oxidative stress, are considered to be closely related to the pathogenesis of AD (3). The deposition of A in the neocortex of overexpressing transgenic mice is accompanied by oxidative damage (4).

However, the biochemistry linking A pathology to oxidative damage in AD is still unclear. We have recently found that the neurotoxicity of A is mediated by the cell-free generation of H₂O₂ by the peptide when it binds catalytic amounts of Cu²⁺ (5). This happens because A is strongly redox-active, reducing Cu²⁺ and Fe³⁺ to Cu⁺ and Fe²⁺, respectively, and subsequently recruiting O₂ as the substrate for H₂O₂ formation (6). The rank order for toxicity, metal reduction, and H₂O₂ formation is A1-42 > A1-40 rat A1-40 (5), in concordance with the participation of the respective peptide in amyloid pathology (2, 7). Since Cu²⁺ and Fe³⁺ are enriched in AD neuropil, and especially in plaque deposits (8), and since H₂O₂ is a freely permeable substrate for oxidation reactions, our findings support the possibility that A interaction with redox active metal ions is a significant source of oxidative stress in AD.

Despite being a striking feature of AD neuropathology, there is no evidence to link histological plaque amyloid directly to cell demise in AD. Amyloid plaque load has been reported not to correlate with the progression of the dementia (9), possibly because there is no correlation between total A load in neocortex and histological plaque count (10). Conversely, a significant correlation is observed between neurofibrillary tangles and neuritic changes, and elevated levels of soluble A in the AD-affected neocortex (10). Indeed, modified soluble forms of A1-42 extracted from AD brain have been shown to possess enhanced toxicity (11). These observations suggest that plaque amyloid may represent a fraction of total A in the brain that has been condensed and neutralized and no longer contributes to neurotoxicity. Therefore the neurochemical factors responsible for condensing A into amyloid are important to identify, since they may represent an effective tissue protective response.

Zn²⁺ is also significantly increased in the AD-affected neocortex and greatly enriched in both human (12) and transgenic mouse -amyloid plaque cores (13). A possesses a high (100 nM) affinity binding site that is very highly specific for Zn²⁺ and a low (5 µM) affinity Zn²⁺ binding site of less selectivity that mediates protease resistance and precipitation of the peptide into amyloid (14, 15). Zn²⁺-mediated assembly of A is reversible with chelation (16), which appears to be the mechanism by which chelators increase the extraction of A from postmortem AD-affected brain samples (17). Zn²⁺ is a redox-inert antioxidant, and recently we reported that coincubation of A with Zn²⁺ inhibits Cu²⁺ reduction (6), therefore we hypothesized that Zn²⁺ may also inhibit H₂O₂ production from A. Here we report that Zn²⁺ inhibits A1-42 neurotoxicity in cell culture, correlating with a suppression of H₂O₂ generated from the Cu²⁺ interaction with the peptide, and a survey of AD-affected histological brain sections revealed that there is an inverse correlation between plaque deposits and 8-OH guanosine (8-OHG) levels in AD-affected brain tissue. These findings suggest that amyloid plaques in AD may form as a result of a more robust tissue zinc response, representing the effective quenching of abnormal A-mediated redox activity.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Reagents-- All chemicals used in this study were culture tested and of high grade and purity (purchased from Sigma, unless specified otherwise). Synthetic human A1-42 peptide was obtained from Yale University, W. M. Keck Foundation Biotechnology Resource Laboratory (Batch numbers SP869BUS, SP902BUS, SP904BUS, SP919BUS, SP956BUS), dissolved in sterile distilled-deionized water, sonicated (3 min), and spun at 13,000 × g (15 min). The supernatant was used for cultures and in vitro assays.

Cell Culture-- The protocol (number 96-4159) used in this study was approved under guidelines for animal research (Massachusetts General Hospital). Neuronal cultures were taken from frontal cortices of Harlan Sprague-Dawley rat embryonic (E18) pups. The cells were dissociated in Hanks' buffered saline solution (Life Technologies, Inc.) with 50 µM kynurenic acid, 10 mM pyruvate, and 100 mM HEPES, and plated (5 × 10⁵ cells/well) in a 12-well polyethyleneimine-coated plate (Corning). Cortical cultures were grown at 95% O₂, 5% CO₂, 85% humidity for 5 days in serum-free Neurobasal medium with B-27 supplement (Life Technologies, Inc.), 20 µM L-glutamate, 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM L-glutamine. On the 5th day (treatment day), the medium was replaced with serum-free Neurobasal plus L-glutamine without B-27 supplement, since B-27 contains various antioxidants that would mask the effects being investigated. Stock solutions were mixed in vehicle medium to final concentrations in the cell culture of 20 µM A1-42, 20 µM ZnCl₂, 20 µM CuCl₂, 10 µM buthionine sulfoximine (BSO), or 1000 units/ml catalase. Experimental trials were done in triplicate wells. Viable cells stained with calcein-AM (Molecular Probes) were assessed using an automated cell counter or counted manually.

In a separate series of experiments, human embryonic kidney 293 cells were cultured in Dulbecco's minimum essential medium (DMEM) with 10% fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin. The day before treatment, the cells were plated in triplicates in a 24-well uncoated plate (125,000 cells/well). The cells were treated with A1-42 with or without ZnCl₂ in serum-free DMEM with L-glutamine, penicillin, and streptomycin. The CellTiter 96® nonradioactive cell proliferation assay (Promega) was used to assess cell viability 72 h following treatment. The wells were washed with phenol red-free, serum-free DMEM prior to addition of the sterile tetrazolium dye (15:100 dye ratio). Negative control was treated with 0.1% Triton X-100 before addition of the dye. Then, the plate was returned to the 37 °C incubator and left for 4 h. Following incubation, the solubilization/stop solution (500 µl) was added and was left for another hour to allow formazan crystals to solubilize. The final reaction product was read at 570 nm.

Data were analyzed using one-way analysis of variance followed by a post-hoc Student's t test. Significance level was set at p < 0.05.

Hydrogen Peroxide Assay-- The cell-free H₂O₂ assay was performed on triplicate samples using a 96-well microtiter plate (SpectraMax Plus, Molecular Devices). A1-42 peptide (10 µM), ZnCl₂ (1 or 10 µM) or CuCl₂ (1 or 10 µM), and a H₂O₂-scavenging agent tris(2-carboxyethyl)phosphine hydrochloride (TCEP; Pierce, 50 µM), were coincubated in Dulbecco's PBS buffer (300 µl), pH 7.4, for 1 h at 37 °C. Following incubation, the amount of H₂O₂ produced was deduced by measuring unreacted TCEP, according to our recently published protocol (6).

Brain Histopathology-- Cerebral cortex was obtained from 22 AD cases (ages 57-93 years, average 78.2) with postmortem intervals of 2-22 h (average 5.8) and prepared as described previously (18). Relative scale measurements of 8-OHG (immunocytochemistry using monoclonal antibody 1F7, which detects both DNA-associated 8-OH-2'-deoxyguanosine and RNA-associated 8-OH-2'-guanosine), and the area of A deposition (immunocytochemistry using monoclonal antibody 4G8, which detects A residues 17-24, Senetek), were performed using a Quantimet 570C Image Processing and Analysis system (Leica) as described previously (18). The anti-A antibody (4G8) recognizes senile plaques of various morphologies, as well as vessel-associated amyloid. The computer analysis followed the protocols of Hyman et al. (9) and involved identifying plaques by gray scale thresholding of immunoreactive deposits, and manually deleting the vessel-associated amyloid, as well as artifacts in the captured image, prior to measurement. There was no significant relationship between postmortem interval and 8-OHG levels in these samples.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Zn²⁺ Inhibits A1-42 Cytotoxicity-- Since H₂O₂ mediates the enhancement of A toxicity by Cu²⁺ (5), we examined the modulation of A toxicity in primary rat brain cultures by Zn²⁺ and Cu²⁺ and contrasted this with the effects of coincubating A with either a H₂O₂ scavenger (catalase, 1000 units/ml) or BSO (10 µM). By inhibiting -glutamylcysteine synthetase, BSO inhibits the synthesis of glutathione, the major intracellular H₂O₂ scavenger (19). We found that 38% of cells treated with freshly prepared A1-42 alone (20 µM) survived after 48 h incubation (Fig. 1A). Catalase protected neurons against A1-42 neurotoxicity (60% survival) in agreement with previous findings (5), while BSO exacerbated the cytotoxicity of A (7% survival, Fig. 1A), consistent with a role for H₂O₂ in mediating A toxicity.

View larger version (21K): [in this window] [in a new window]   Fig. 1.   Effect of Zn2+ upon A1-42 cytotoxicity. A and B, primary rat neuronal cultures were incubated with A1-42 (A, 20 µM; B, 10 µM) and/or other factors for 48 h, and cell survival measured by live-dead assay, compared with untreated cultures. A, effects of coincubation with catalase (A+Cat), buthionine sulfoximine (A+BSO), Zn2+ (20 µM, A+Zn), Cu2+ (20 µM, A+Cu), and effects of these factors alone, upon neuronal survival are shown. C, human embryonic kidney 293 cells were incubated with A1-42 (10 µM) ± Zn2+ (10 µM) or Zn2+ alone, as shown. Surviving cells were assayed, compared with untreated control cultures. Data are means ± S.E., n = 5-6 experimental trials performed in triplicate (*, p < 0.01; **, p < 0.001).

Paralleling the effect of catalase upon cell survival, the presence of Zn²⁺ (1:1 with A, 20 µM) significantly protected against A1-42 neurotoxicity (62% survival). In agreement with our previous findings (5), Cu²⁺ significantly exaggerated the cytotoxicity of A1-42 (18% survival), which paralleled the effects of BSO. The effect of Zn²⁺ upon inhibiting A1-42 toxicity was similar at a lower concentration (10 µM, A:Zn²⁺ = 1:1, not shown). The presence of catalase, BSO, Cu²⁺, or Zn²⁺ alone had no significant effect on cell survival compared with untreated cells, suggesting that the effects of Cu²⁺ and Zn²⁺ in modulating A toxicity were due to interaction with the peptide.

We also observed that Cu²⁺ and Zn²⁺ had competitive effects upon A1-42 toxicity in primary cortical cultures when coincubated at equimolar ratios (1:1:1) (Fig. 1B). To confirm that the rescue of A toxicity by Zn²⁺ was due to interaction with the peptide and not due to Zn²⁺ binding to cellular elements in the mixed brain culture (e.g. inhibiting an excitotoxic response by binding to the NMDA receptor), we studied the effects of Zn²⁺ (1:1) upon A toxicity in human embryonic kidney 293 cell culture. Similar to the rescue of A toxicity in cortical primary cell culture (Fig. 1A), we found that Zn²⁺ also inhibited the toxicity of A upon the kidney cells (Fig. 1C).

Zn²⁺ Inhibits H₂O₂ Production from A1-42-- To explore whether the inhibition of A toxicity by the presence of Zn²⁺ was due to the quenching of H₂O₂ production from A, we studied the effect of Zn²⁺ on the generation of H₂O₂ by A1-42 in a cell-free system. A1-42 (10 µM) was coincubated with Cu²⁺ (1 µM), since we have previously shown that H₂O₂ production by A depends upon the presence of substoichiometric amounts of coper or iron (6). In the presence of 1 µM copper, A1-42 produced 4.9 µM H₂O_2. (Fig. 2). Coincubation of this mixture with an equimolar concentration of ZnCl₂ (1 µM) significantly decreased H₂O₂ production by 25%, while coincubation with 10-fold excess ZnCl₂ (10 µM) markedly inhibited H₂O₂ production by 90%. The presence of the metal ions alone promoted no significant H₂O₂ production in the absence of A (Fig. 2).

View larger version (11K): [in this window] [in a new window]   Fig. 2.   Effect of Zn2+ on cell-free H2O2 production by A1-42. A1-42 peptide (10 µM) was incubated for 1 h at 37 °C in PBS with CuCl2 (copper 1 (Cu1) = 1 µM), ± ZnCl2 (zinc 1 (Zn1) = 1 µM, zinc 10 (Zn10) = 10 µM) and levels of H2O2 measured. The background levels of H2O2 production in the absence of peptide were also measured. Data are means ± S.E. (n = 5 experimental trials performed in triplicate wells).

Since Zn²⁺ is concentrated in plaque cores to 1 mM (8, 12, 13), we hypothesized that plaque may represent A that does not form H₂O₂, and hence the abundance of plaque cores would not be expected to correlate with oxidation damage in AD. Therefore, we surveyed the relationship between tissue oxidation and amyloid density in AD postmortem tissue.

Inverse Correlation between Plaque Density and Oxidation in AD-- To test the hypothesis that amyloid deposits represent the fraction of A that has been successfully neutralized by Zn²⁺ in AD, we performed a correlative analysis between the quantity of histological amyloid and parenchymal oxidative damage in AD affected neocortical tissue. This revealed that there was a highly significant, log-linear inverse correlation between A levels and oxidative damage to nucleic acid (8-OHG) (Fig. 3), suggesting that the formation of amyloid plaque is associated with tissue protection in vivo. There was no obvious decrease in the 8-OHG intensity in neurons based on their apparent proximity to plaques, in agreement with our previous report (18).

View larger version (17K): [in this window] [in a new window]   Fig. 3.   Inverse correlation between A burden and levels of oxidized nucleic acids (8-OHG) in AD brain. Immunohistochemical levels of A and 8-OHG in postmortem AD (n = 22) cases were quantified by computer-assisted image analysis as described previously (18). %SP = percent immunoreactive senile plaque surface area.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A induces neurotoxicity through cell-free H₂O₂ generation (5), and our current findings indicate that Zn²⁺ binding to A inhibits neurotoxicity through suppressing cell-free H₂O₂ production. We also found that there is an inverse correlation between amyloid plaque density in AD and oxidation of neocortical tissue. Since plaque concentrations of Zn²⁺ are high enough to both precipitate A (14, 15) and suppress H₂O₂ in vitro, it is therefore likely that the Zn²⁺ within the amyloid plaques makes them redox-inert. This interpretation is consistent with the lack of oxidative modifications within the senile plaques such as nitrotyrosine and carbonyl adducts (18, 20) that are found within neurons in the AD-affected neocortex. That we did not find a spatial relationship between 8-OHG and plaques may reflect the rapid migration of RNA (18) throughout the neuron, or alternatively, that the oxidizing stress comes from a ubiquitous origin such as elevated soluble A whose levels correlate with tangle and neuritic pathology, and inversely with life-span, in AD (10). Taken together, these data suggest a protective role for Zn²⁺ in ameliorating A-mediated oxidative damage in AD.

Our findings are in agreement with a recent report that Zn²⁺ attenuates the toxicity of A1-40 and may explain the observed rescue of cellular Na⁺/K⁺ ATPase activity (21). Since Na⁺/K⁺ ATPase is sensitively inhibited by H₂O₂ (22), its rescue by coincubating Zn²⁺ with A may in fact be due to the inhibition of cell-free generation of H₂O₂.

H₂O₂ production by A is dependent upon the reduction of Cu²⁺ or Fe³⁺ by the peptide. We previously found that when incubated at equimolar concentrations with Cu²⁺ in the presence of A1-42, Zn²⁺ inhibits 50% of the production of Cu⁺ (6). This may explain the 25% decrease in cell-free H₂O₂ produced by A1-42 in the presence of equimolar concentrations of Zn²⁺ and Cu²⁺ (1 µM) (Fig. 2), since a proposed mechanism for O₂² formation by A:Cu⁺ involves the donation of one electron from each of two Cu⁺ to O₂ (5, 6). We recently found that when A is coincubated with equal concentrations of Cu²⁺ and Zn²⁺ it binds equimolar amounts of both metal ions (1.5 equivalents of each at pH 7.4; Ref. 23). This suggests that there are separate selective Cu²⁺ and Zn²⁺ binding sites on A and that when inhibiting H₂O₂ production, Zn²⁺ may displace nonspecific Cu²⁺ binding from a redox-active binding site on A.

A1-42 possesses a much higher affinity for Cu²⁺ (high affinity K_d = 10¹⁷ M, low affinity K_d = 10⁸; Ref. 23) compared with its highest affinity for Zn²⁺ (K_d 10⁷; Ref. 14); therefore, in its soluble interstitial form, A1-42 is likely to bind Cu²⁺ before it binds Zn²⁺. Zn²⁺ generally exerts its antioxidant effects by protecting free sulfhydryl groups from oxidation (there are none on A) and by competing with prooxidant metal ions for binding sites and decreasing their ability to transfer electrons pathologically. We therefore propose that A may form amyloid where the levels of interstitial Zn²⁺ are sufficiently elevated to compete with low affinity Cu²⁺ or Fe³⁺ binding to A and so quench the H₂O₂ generated by soluble, neurotoxic forms of A, at the expense of forming amyloid. Conversely, amyloid-poor brain tissue may be more prone to oxidation damage (Fig. 3) mediated by high levels of toxic, soluble A (10), because tissue Zn²⁺ concentrations are insufficiently elevated to form amyloid plaque.

There is a large body of evidence indicating that zinc, copper, and iron are significantly elevated in the AD brain (reviewed in Ref. 3). Whereas abnormal Cu²⁺ elevation may drive the toxicity of A (5), interstitial Zn²⁺ elevation may reflect a homeostatic antioxidant response. Mechanistically, this could be due to Zn²⁺ release from the metallothionein (MT) pool upon glial activation (24), or due to MT thiols being oxidized by H₂O₂ (25). The hypothesis that Zn²⁺ elevation forms amyloid is supported by the distribution of chelatable (loosely bound) Zn²⁺ in the brain, which is most highly concentrated in the corticofugal system (26) and therefore parallels the anatomical sites most prone to amyloid deposition.

Other A-associated proteins may also modulate the precipitation of A in the presence of Zn²⁺, and so play a role in amyloid formation. The Zn²⁺ binding properties of ₂-macroglobulin, a genetic risk factor for AD (27), modulate its binding to A (28). Also, we have recently reported that apolipoprotein E preserves A solubility in the presence of Zn²⁺, and that the ApoE4 isoform, another risk factor for amyloid deposition and AD, is the poorest solubility chaperone under these conditions (29). Therefore, in ApoE4 carriers, A is more likely to be precipitated by Zn²⁺ and, according to our current findings, disqualified from neurotoxic H₂O₂ production. However, ApoE is also an antioxidant that protects against H₂O₂-mediated damage, but ApoE4 is the poorest H₂O₂ antioxidant of the isoforms (30, 31), in concordance with the observation that AD patients with this allele have more oxidative injury to the neocortex than non-ApoE4 carriers (31). Therefore, whereas Zn²⁺-mediated A precipitation may contribute to the increased amyloid plaque burden of ApoE4 carriers with AD, the quenching of A-associated H₂O₂ production brought about by this response appears to be insufficient to compensate for the decreased antioxidant properties of ApoE4.

We previously suspected that Zn²⁺ overload in the brain may be detrimental in the pathogenesis of AD, because Zn²⁺ dramatically produced amyloid from soluble A in vitro (15), and because a pilot clinical study had observed cases where zinc supplementation acutely worsened cognition in AD subjects. Our current findings force us to reconsider the position of Zn²⁺ in the pathophysiology of AD. No clear evidence has emerged that zinc supplementation is deleterious to the progression of AD, and upon further investigation, the detrimental effects of supplementation observed previously may have been due to gastrointestinal disturbance.² However, there also have not been any statistically satisfactory reports of the beneficial clinical effects of Zn²⁺ supplementation upon AD. We recently reported that zinc/copper-selective chelators markedly enhance the resolubilization of A deposits from postmortem AD brain samples (17), supporting the possibility that copper and zinc ions play a significant role in assembling amyloid. If a chelation approach were to be translated into an in vivo therapy for AD, our current findings indicate that it would be necessary to consider the risk of increasing brain oxidation damage if Zn²⁺ were removed from the amyloid mass before the removal of Cu²⁺ and Fe³⁺.

	FOOTNOTES

^* This work was supported by funds from the American Health Assistance Foundation, Prana Corp., Alzheimer's Association, by National Institute on Aging Grants R29-12686 and RO1-AG09287, and by the NH&MRC.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ Recipient of the New Zealand Neurological Foundation Miller Postgraduate Scholarship.

To whom correspondence should be addressed: Director, Laboratory for Oxidation Biology, Massachusetts General Hospital, Bldg. 149, 13th St., Charlestown, MA 02129. Tel.: 617-726-8244; Fax: 617-724-9610; E-mail: bush@helix.mgh.harvard.edu.

Published, JBC Papers in Press, May 8, 2000, DOI 10.1074/jbc.C000165200

² A. I. Bush and C. L. Masters, personal communication.

	ABBREVIATIONS

The abbreviations used are: AD, Alzheimer's disease; 8-OHG, 8-OH guanosine; BSO, buthionine sulfoximine; DMEM, Dulbecco's minimum essential medium; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; PBS, phosphate-buffered saline; MT, metallothionein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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				R. Squitti, D. Lupoi, P. Pasqualetti, G. Dal Forno, F. Vernieri, P. Chiovenda, L. Rossi, M. Cortesi, E. Cassetta, and P. M. Rossini Elevation of serum copper levels in Alzheimer's disease Neurology, October 22, 2002; 59(8): 1153 - 1161. [Abstract] [Full Text] [PDF]

				C. Opazo, X. Huang, R. A. Cherny, R. D. Moir, A. E. Roher, A. R. White, R. Cappai, C. L. Masters, R. E. Tanzi, N. C. Inestrosa, et al. Metalloenzyme-like Activity of Alzheimer's Disease beta -Amyloid. Cu-DEPENDENT CATALYTIC CONVERSION OF DOPAMINE, CHOLESTEROL, AND BIOLOGICAL REDUCING AGENTS TO NEUROTOXIC H2O2 J. Biol. Chem., October 18, 2002; 277(43): 40302 - 40308. [Abstract] [Full Text] [PDF]

				M. H. Mohajeri, K. Saini, J. G. Schultz, M. A. Wollmer, C. Hock, and R. M. Nitsch Passive Immunization against beta -Amyloid Peptide Protects Central Nervous System (CNS) Neurons from Increased Vulnerability Associated with an Alzheimer's Disease-causing Mutation J. Biol. Chem., August 30, 2002; 277(36): 33012 - 33017. [Abstract] [Full Text] [PDF]

				K. Zou, J.-S. Gong, K. Yanagisawa, and M. Michikawa A Novel Function of Monomeric Amyloid beta -Protein Serving as an Antioxidant Molecule against Metal-Induced Oxidative Damage J. Neurosci., June 15, 2002; 22(12): 4833 - 4841. [Abstract] [Full Text] [PDF]

				A. I. Bush and R. E. Tanzi The galvanization of beta -amyloid in Alzheimer's disease PNAS, May 28, 2002; 99(11): 7317 - 7319. [Full Text] [PDF]

				S. Melov, S. R. Doctrow, J. A. Schneider, J. Haberson, M. Patel, P. E. Coskun, K. Huffman, D. C. Wallace, and B. Malfroy Lifespan Extension and Rescue of Spongiform Encephalopathy in Superoxide Dismutase 2 Nullizygous Mice Treated with Superoxide Dismutase-Catalase Mimetics J. Neurosci., November 1, 2001; 21(21): 8348 - 8353. [Abstract] [Full Text] [PDF]

				F. W. Sunderman Jr. Nasal Toxicity, Carcinogenicity, and Olfactory Uptake of Metals Ann. Clin. Lab. Sci., January 1, 2001; 31(1): 3 - 24. [Abstract] [Full Text] [PDF]

				Y. Yoshiike, K. Tanemura, O. Murayama, T. Akagi, M. Murayama, S. Sato, X. Sun, N. Tanaka, and A. Takashima New Insights on How Metals Disrupt Amyloid beta -Aggregation and Their Effects on Amyloid-beta Cytotoxicity J. Biol. Chem., August 17, 2001; 276(34): 32293 - 32299. [Abstract] [Full Text] [PDF]