Coping with Copper—Minute Amount of Metal Plagues Rabbit Brain

Copper has long been implicated as a contributory factor in the development of Alzheimer's disease, but the role of this divalent cation remains controversial. In this week's PNAS early online edition, Larry Sparks, Sun Health Research Institute, Sun City, Arizona, and Bernard Schreurs, West Virginia University, Morgantown, report that, depending on the diet, even minor amounts of the metal can lead to both amyloid plaques and a memory deficit in rabbits.

The diet to elicit these effects, it turns out, is one supplemented with two percent cholesterol. When Sparks fed rabbits such a cholesterol diet for 10 weeks, then examined various regions of the brain, he found that the animals had almost twice as many Aβ-immunoreactive neurons in the cortex as did rabbits spared the cholesterol. In the temporal cortex, for example, cholesterol-fed rabbits had, on average, 108 such neurons per square mm, vs. 58 in rabbits fed normal chow.

Copper exacerbated this difference. When Sparks added 0.12 ppm copper sulfate to the distilled water supply, the number of immunoreactive neurons almost doubled again, to 174/sq mm. Similar effects were seen in the superior cortex (176/sq mm, 98/sq mm, and 54/sq mm, for cholesterol/copper, cholesterol, and normal diet, respectively), and the hilus, while about a 1.5-fold increase was observed in the hippocampus. All these increases were statistically significant. However, the metal had very little effect on neurons in the parietal cortex. The antibody used, 10D5, which reacts with the first 16 amino acids in the N-terminal of Aβ, also revealed some extracellular deposits, or senile plaque-like structures, in the copper-fed animals, but not in the controls.

The metal also interfered with the rabbits' ability to learn a conditioned response. Sparks tested this by exposing the animals to an audible tone that was followed half a second later with a small blast of air. After eight sessions of conditioning, control rabbits anticipated the air puff about 60 percent of the time; rabbits fed copper, however, were much less successful, reacting before the air puff only 10 percent of the time.

This is not the first time copper has been linked to cholesterol and Aβ metabolism. Low density lipoprotein, a cholesterol carrier, has been shown to exacerbate the neurotoxic effect of copper-laced AβPP (see ARF related news story), while the copper chelator clioquinol has been shown to reduce amyloid burden in the brain (see ARF related news story). What's striking about this recent study is the minute levels of copper needed to exacerbate the effects of cholesterol-about 10-fold less than the 1.3 ppm limit for drinking water (U.S.) set by the Environmental Protection Agency.

What all this means for humans is uncertain. It is known that in certain cases, such as the copper transport disease tricholipodystrophy, otherwise known as Menke's or kinky hair disease, grossly elevated copper levels do not lead to dementia. "But it is a two-stage process," explains Sparks. "Copper itself may have no effect unless there is an overproduction of Aβ induced by cholesterol."—Tom Fagan

Comments

  1. The PNAS paper is a follow-up of a similar study published in JAD (Vol 4, 523)in which a component of normal tapwater was suggested to contribute towards cholesterol-induced AD-like pathology. The critical aspect of both papers is that this 'component' was found to potentiate the effects attributed to the presence of additional cholesterol in the rabbit chow. The authors, and indeed those commenting on this work in Science and elsewhere are premature in attributing this potentiation directly to copper. Either of the papers neither demonstrate an increase in systemic cholesterol nor do they show any changes in copper homeostasis. (Why were these analyses not carried out !?) No mention is made of how much copper or cholesterol was already present in the rabbit chow. No mention is made of how the additional cholesterol was incorporated in the diet nor was any information given on how the pH of the drinking water was controlled (distilled water + copper sulphate will be acidic). In addition we do not know whether the amount of cholesterol-supplemented feed that was eaten by the rabbits in the different groups was the same! There are so many imponderables that it is quite amazing that this paper has survived peer review in its present form.

    The points that I have raised might seem trivial but I can assure you that they are not. It seems extremely likely that cholesterol in the diet can contribute towards some forms of AD-like neuropathology. The experiments from the group of Sparks have been important in reaffirming this and in demonstrating that the influence of cholesterol is potentiated by other factors. However, to say that the PNAS paper has identified trace amounts of copper as the critical factor is "gilding the lily" somewhat. Certainly, drinking water quality is influencing the role of cholesterol but we are not given any useful information as to whether this is due to changes in (i) the absorption of cholesterol in the gut; (ii) the absorption of something else from the feed which is influenced by additional cholesterol in the feed; (iii) whether rabbits on some treatments actually ate more cholesetrol-supplemeted diet than those on other feeds; (iv) whether other individual factors which would also alter the chemistry of distilled water might also reproduce the so-called copper effect, ie. no positive control. I am already going on and on and I could continue!!

    Needless to say this paper should not have been published in its current format, which includes an extremely misleading title that has been grasped and expanded upon by an all too eager scientific press. If I had to put my neck on the line, which I will, I will say that the potentiation of the cholesterol effect by copper added to distilled water is nothing to do with any direct (or even specific ?) influence of systemic copper. At best, more biologically available copper in the drinking water might be facilitating the cholesterol effect in the gut but even this is highly unlikely.

    View all comments by Chris Exley

  2. Coping with Copper—Minute Amount of Metal Plaques in Rabbit Brain
    The article by Sparks and Schreurs provides evidence that copper is the water contaminant responsible for increased neuronal and extracellular accumulation of amyloid previously reported by these workers in rabbits (Sparks et al., 2002). These are intriguing observations, given the well-characterized interaction of copper with Aβ (Atwood et al., 1998; Dong et al., 2003), and while the exact biochemical interaction among these molecules (copper, Aβ and cholesterol) remains to be determined, it is clear that cholesterol and copper play an important role in amyloid deposition.

    The relevance of these neuropathological observations to humans is indicated by the identification of cognitive deficits in copper-treated animals. That these cognitive and neuropathological changes occur at concentrations of copper 10 times lower (0.12 ppm, 0.12 mg/liter) than the EPA maximum allowable level suggests that corrosion of copper plumbing (promoted by acidic conditions) may promote amyloid deposition (and cognitive defects). However, this has to be reconciled with the fact that, on average, humans ingest 2-5 mg of copper per day. The consumption of three liters of water a day would only add another 0.36 mg to our diet. However, coupled with a high-cholesterol diet, such an increase may be sufficient to induce amyloid depostion and cognitive changes in humans, as is the case with rabbits in the present study.

    References
    Atwood CS, Moir RD, Huang X, Scarpa RC, Bacarra NM, Romano DM, Hartshorn MA, Tanzi RE, Bush AI. Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem. 1998 May 22;273(21):12817-26. Abstract

    Dong J, Atwood CS, Anderson VE, Siedlak SL, Smith MA, Perry G, Carey PR. Metal Binding and Oxidation of Amyloid-beta within Isolated Senile Plaque Cores: Raman Microscopic Evidence. Biochemistry. 2003 Mar 18;42(10):2768-73. Abstract)

    Sparks DL, Lochhead J, Horstman D, Wagoner T, Martin T. Water quality has a pronounced effect on cholesterol-induced accumulation of Alzheimer amyloid beta (Abeta) in rabbit brain. J Alzheimers Dis. 2002 Dec ;4(6):519-25. Abstract

    References:

    Atwood CS, Moir RD, Huang X, Scarpa RC, Bacarra NM, Romano DM, Hartshorn MA, Tanzi RE, Bush AI. Dramatic aggregation of Alzheimer abeta by Cu(II) is induced by conditions representing physiological acidosis. J Biol Chem. 1998 May 22;273(21):12817-26. PubMed.

    Dong J, Atwood CS, Anderson VE, Siedlak SL, Smith MA, Perry G, Carey PR. Metal binding and oxidation of amyloid-beta within isolated senile plaque cores: Raman microscopic evidence. Biochemistry. 2003 Mar 18;42(10):2768-73. PubMed.

    Sparks DL, Lochhead J, Horstman D, Wagoner T, Martin T. Water quality has a pronounced effect on cholesterol-induced accumulation of Alzheimer amyloid beta (Abeta) in rabbit brain. J Alzheimers Dis. 2002 Dec;4(6):523-9. PubMed.

    View all comments by Craig Atwood

  3. I have to agree completely with the comments of Exley. The illustrations purporting to demonstrate senile plaques are not convincing. Antibody immunoreactivity seems greater in some sets of animal than others, but without actual measurements of copper levels, it is hard to decide whether copper has anything to do with the increased antibody reactivity. Measuring enzyme levels as surrogate markers of metal levels are quite inadequate. I can't judge the significance of the animal conditioning data, but I don't believe the this rabbit model has been characterized well enough to take them seriously.
    The claim that low levels of copper and cholesterol feeding are synergistic in enhancing Abeta production and amyloid plaque formation in rabbits implies a cause and effect relationship which remains unproven, and the implications of these findings, from a public health point of view, are too important to rest on such inadequate data.

    View all comments by Vincent Marchesi

  4. I think that copper is a neurotoxic substance that displaces the zinc in the cell-specific carbonic anhydrase enzymes in the brain, leading
    to their death and to the production of amyloid plaques. Cell-specific carbonic anhydrase enzymes in the brain produces hydrogen ions
    which serve as the fuel of the ion pump that maintains the integrity of the cell membrane. Depolarization of the cell membrane causes the influx of water, Na+, Ca++, and other neurotoxic materials, such as aluminum, lead, and iron, that displace the zinc from the cell-specific carbonic anhydrase enzymes—cellular death follows. Decreased levels of cell-specific carbonic anhydrase lead to cell death.

    For references see WIPO publication #WO 03/070167 A2; I am the author.

    References:
    World Intellectual Property Organization (WIPO)
    publication #WO 03/070167 A2—published on
    August 28, 2003. "Therapeutic and Prophylactic
    Treatment of Aging and Disorders of Aging which
    includes Alzheimer's Disease."

    View all comments by Victorio Rodriguez

  5. Copper: A Role in AD?
    Recent exciting findings suggest that copper in drinking water is able to exacerbate the amyloid pathology and an associated learning deficit in the cholesterol-fed rabbit model of Alzheimer's disease (Sparks et al., 2002; Sparks and Schreurs, 2003). Such data, together with previous studies linking aluminum (Crapper et al., 1973), zinc (Cuajungco et al., 2000), and iron (Smith et al., 1997) to Alzheimer's disease, suggest that metals may play a key role in disease pathogenesis (Perry et al., 2003). However, while aluminum (Pratico et al., 2002), and now copper (Sparks et al., 2002; Sparks and Schreurs, 2003), increase amyloid pathology and chelation therapy reduces amyloid burden (Cherny et al., 2001) in animal models, whether similar mechanisms are at play in human patients is still under investigation. Additionally, while one could simply equate these findings with the notion that metal-induced alterations in amyloid leads to amyloid-induced neuronal damage, an equally logical interpretation would be that metal-induced neuronal damage results in a compensatory increase in amyloid to provide neuroprotection (Smith et al., 2002; Rottkamp et al., 2002). Needless to say, whatever the mechanism involved, and despite our utmost respect for the investigators involved, we feel it is somewhat premature at this point in time to begin replacing copper plumbing from our homes with glass pipes!

    References:

    Cherny RA, Atwood CS, Xilinas ME, Gray DN, Jones WD, McLean CA, Barnham KJ, Volitakis I, Fraser FW, Kim Y, Huang X, Goldstein LE, Moir RD, Lim JT, Beyreuther K, Zheng H, Tanzi RE, Masters CL, Bush AI. Treatment with a copper-zinc chelator markedly and rapidly inhibits beta-amyloid accumulation in Alzheimer's disease transgenic mice. Neuron. 2001 Jun;30(3):665-76. PubMed.

    Crapper DR, Krishnan SS, Dalton AJ. Brain aluminum distribution in Alzheimer's disease and experimental neurofibrillary degeneration. Science. 1973 May 4;180(4085):511-3. PubMed.

    Cuajungco MP, Goldstein LE, Nunomura A, Smith MA, Lim JT, Atwood CS, Huang X, Farrag YW, Perry G, Bush AI. Evidence that the beta-amyloid plaques of Alzheimer's disease represent the redox-silencing and entombment of abeta by zinc. J Biol Chem. 2000 Jun 30;275(26):19439-42. PubMed.

    Perry G, Taddeo MA, Petersen RB, Castellani RJ, Harris PL, Siedlak SL, Cash AD, Liu Q, Nunomura A, Atwood CS, Smith MA. Adventiously-bound redox active iron and copper are at the center of oxidative damage in Alzheimer disease. Biometals. 2003 Mar;16(1):77-81. PubMed.

    Praticò D, Uryu K, Sung S, Tang S, Trojanowski JQ, Lee VM. Aluminum modulates brain amyloidosis through oxidative stress in APP transgenic mice. FASEB J. 2002 Jul;16(9):1138-40. PubMed.

    Rottkamp CA, Atwood CS, Joseph JA, Nunomura A, Perry G, Smith MA. The state versus amyloid-beta: the trial of the most wanted criminal in Alzheimer disease. Peptides. 2002 Jul;23(7):1333-41. PubMed.

    Smith MA, Casadesus G, Joseph JA, Perry G. Amyloid-beta and tau serve antioxidant functions in the aging and Alzheimer brain. Free Radic Biol Med. 2002 Nov 1;33(9):1194-9. PubMed.

    Smith MA, Harris PL, Sayre LM, Perry G. Iron accumulation in Alzheimer disease is a source of redox-generated free radicals. Proc Natl Acad Sci U S A. 1997 Sep 2;94(18):9866-8. PubMed.

    Sparks DL, Schreurs BG. Trace amounts of copper in water induce beta-amyloid plaques and learning deficits in a rabbit model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):11065-9. PubMed.

    Sparks DL, Lochhead J, Horstman D, Wagoner T, Martin T. Water quality has a pronounced effect on cholesterol-induced accumulation of Alzheimer amyloid beta (Abeta) in rabbit brain. J Alzheimers Dis. 2002 Dec;4(6):523-9. PubMed.

    View all comments by Craig Atwood

  6. Comment by Rebecca J. Henderson and James R. Connor
    Much attention has been paid to the link between AD and metal ions. These studies go back to the imbalance of iron found in the brain in AD and the contribution of iron to oxidative stress [1], and even earlier to the idea that aluminum toxicity was involved in the pathogenesis of AD. More recently, data have been presented indicating that β-amyloid has a relatively high binding affinity for zinc, iron, and copper. Metal complexing agents are under investigation as therapeutic agents in Alzheimer’s disease [2,3]. Because metals are acquired through dietary and environmental sources, one mechanism by which metal availability could be manipulated is through the diet. Three recent papers published in PNAS attempt to elucidate more clearly copper’s effect, if any, on the disease state. Two of the papers [4,5] propose beneficial actions for copper, while work by Sparks and Schruers [6] claims that dietary copper exacerbates the disease.

    Phinney et al. use a potentially powerful technique of crossing two transgenic animal models in order to evaluate how a propensity for higher uptake of copper may impact the deposition of amyloid. The result was an unexpected decrease in plaque burden in the animals with the mutation that should increase copper levels. The data are promising and reveal important areas for future study, but have some limitations. In these types of studies it is important to show that the changes in the amount of total brain copper or any metal are occurring in the same regions as alterations in amyloid expression, processing or plaque burden. It is difficult to reconcile, for example, how the lack of any change in copper concentration at two months of age (Figure 2) can be directly related to a change in Aβ (Figure 5) when the combined transgenic animals have an increase in brain copper but no difference in the Aβ brain concentrations (Figures 3b and d). The plasma decreases of Aβ noted in the study are interesting and worthy of pursuit. However, at this time a relationship between plasma Aβ and brain Aβ has not been established. The authors attempt to address the concern about the distribution of copper and plaques by providing the data in Figure 4. However, in order to interpret these data as directly relevant to copper, the concentrations of copper in the hippocampus must be determined. Nonetheless, the authors have shown that elevations in total brain copper do not increase plaque burden or Aβ levels in brain, which warrants further investigation.

    Bayer et al. [5] provide provocative data of a possible sex-linked difference in response to dietary copper. Their data illustrating a reduction in lethality after copper administration is impressive. However, the increase of brain copper levels the researchers aimed to achieve was barely significant above control levels. Furthermore, as in the Phinney et al. study, the regional levels of copper and changes in the other parameters are critical to understanding any potential relationship. The substantial error bars make the data in this study difficult to evaluate.

    The final PNAS paper on which we are offering comment found that trace amounts of copper in the drinking water can increase markers of AD in the brain in cholesterol-fed rabbits [6]. Although in apparent contrast to the previous two papers, it must be remembered that these rabbits were cholesterol-fed. There is no control group for copper without cholesterol, so the direct contribution of copper is not clear. A significant experimental design concern with this study is the amount of copper administered. As mentioned in the paper, 0.9 mg./day of copper is the EPA’s normal tolerable upper limit for the metal. The rabbits in this study consumed between 0.04 and 0.08 mg./day based on average amount of water ingested. If the rabbits weighed 2.2 kg., this would be equivalent to a 1.2 to 2.4 mg./day dose of copper for a 150 lb. human, well above the EPA limit. Therefore, the applicability of this study to humans exposed to normal levels of environmental copper is questionable, as is the relationship to AD pathogenesis. High cholesterol, and high copper plus high cholesterol could induce AD-like morphological changes in the brain, whereas according to the previous two studies, high copper alone may actually be protective. Therefore, in the context of the previous papers, the data from the study by Sparks and Schreurs could be interpreted to indicate that decreasing cholesterol should be the goal in a copper-rich environment.

    These studies underscore the importance of an environmental or dietary factor in the induction of AD-like pathology in the brain and are important, given the low number of genetic mutations associated with AD. The data offer compelling evidence that investigations into the contribution of biometals to AD and clinical studies involving metal chelation therapy in AD are worthy of support.

    See also:

    Connor, J.R., ed. Metals and Oxidative Damage in Neurological Disorders. 1997, Plenum Press: New York.

    Malecki, E.A. and J.R. Connor, The case for iron chelation and/or antioxidant therapy in Alzheimer's disease. Drug Development Research, 2002. 51: p. 1-5.

    References:

    Bush AI. Copper, zinc, and the metallobiology of Alzheimer disease. Alzheimer Dis Assoc Disord. 2003 Jul-Sep;17(3):147-50. PubMed.

    Phinney AL, Drisaldi B, Schmidt SD, Lugowski S, Coronado V, Liang Y, Horne P, Yang J, Sekoulidis J, Coomaraswamy J, Chishti MA, Cox DW, Mathews PM, Nixon RA, Carlson GA, St George-Hyslop P, Westaway D. In vivo reduction of amyloid-beta by a mutant copper transporter. Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14193-8. PubMed.

    Bayer TA, Schäfer S, Simons A, Kemmling A, Kamer T, Tepest R, Eckert A, Schüssel K, Eikenberg O, Sturchler-Pierrat C, Abramowski D, Staufenbiel M, Multhaup G. Dietary Cu stabilizes brain superoxide dismutase 1 activity and reduces amyloid Abeta production in APP23 transgenic mice. Proc Natl Acad Sci U S A. 2003 Nov 25;100(24):14187-92. PubMed.

    Sparks DL, Schreurs BG. Trace amounts of copper in water induce beta-amyloid plaques and learning deficits in a rabbit model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):11065-9. PubMed.

    View all comments by Rebecca J. Henderson

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References

News Citations

  1. Good Copper, Bad Copper: News on this Metal's Role in Alzheimer's
  2. Two Ways to Attack Amyloid: Metal Chelator and Antibody

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Primary Papers

  1. Sparks DL, Schreurs BG. Trace amounts of copper in water induce beta-amyloid plaques and learning deficits in a rabbit model of Alzheimer's disease. Proc Natl Acad Sci U S A. 2003 Sep 16;100(19):11065-9. PubMed.

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