Intracellular Copper Levels Research Articles

SHORT COMMUNICATION: Functional consequences of RNA interference targeting COMMD1 in a canine hepatic cell line in relation to copper toxicosis

SummaryA deletion in the copper metabolism (Murr1) domain containing 1 (COMMD1) gene is associated with hepatic copper toxicosis in dogs, yet evidence of copper retention in COMMD1‐depleted hepatic cells has not been shown. In a dog hepatic cell line, we analysed the copper metabolic functions after an 80% (mRNA and protein) COMMD1 reduction with COMMD1‐targeting siRNAs. Exposure to 64Cu resulted in a significant increase in copper retention in COMMD1‐depleted cells. COMMD1‐depleted cells were almost three times more sensitive to high extracellular copper concentrations. Copper‐mediated regulation of metallothionein gene expression was enhanced in COMMD1‐depleted cells. Based on the increased copper accumulation and enhanced cellular copper responses upon COMMD1 reduction, we conclude that COMMD1 has a major regulatory function for intracellular copper levels in hepatic cells.

Regulation of ceruloplasmin in human hepatic cells by redox active copper: identification of a novel AP-1 site in the ceruloplasmin gene

Cp (ceruloplasmin), a copper containing plasma protein, mainly synthesized in the liver, is known to be functional between the interface of iron and copper metabolism. We have reported previously that Cp is regulated by cellular iron status, but the process of the regulation of Cp by copper still remains a subject for investigation. In the present paper, we show that PDTC (pyrrolidine dithiocarbamate), a thiol compound widely known to increase intracellular redox copper, regulates Cp expression in hepatic cells by a copper-dependent transcriptional mechanism. To find out the mechanism of induction, chimeric constructs of the Cp 5'-flanking region driving luciferase were transfected into human hepatic cells. Deletion and mutational analyses showed the requirement of a novel APRE [AP-1 (activator protein-1) responsive element] present about 3.7 kb upstream of the translation initiation site. The role of AP-1 was confirmed by electrophoretic mobility-shift analysis. Western blot and overexpression studies detected the AP-1 as a heterodimer of c-jun and c-fos proteins. The activation of AP-1 was found to be copper-dependent as a specific extracellular chelator bathocuproine disulfonic acid blocked PDTC-mediated induction of AP-1-DNA binding and increased reporter gene activity. Whereas, in a copper-free medium, PDTC failed to activate either AP-1 or Cp synthesis, supplementation of copper could reverse AP-1 activation and Cp synthesis. Our finding is not only the first demonstration of regulation of Cp by redox copper but may also explain previous findings of increased Cp expression in cancers like hepatocarcinoma, where the intracellular copper level is higher in a redox compromised environment.

MDM2: A Novel Mineralocorticoid-Responsive Gene Involved in Aldosterone-Induced Human Vascular Structural Remodeling

Aldosterone has been demonstrated to play an important role in the pathogenesis of various cardiovascular diseases. Vascular structural remodeling, including vascular smooth muscle cell (VSMC) proliferation, has been also reported in small resistance arteries of patients with primary aldosteronism. Therefore, in this study, we examined whether genes involved in the regulation of the cell cycle were induced by aldosterone alone in cultured human VSMCs and in human small resistance arteries. Results of these studies eventually demonstrated that MDM2, one of the genes involved in anti-apoptosis and cell growth, was markedly increased in mineralocorticoid receptor (MR)-positive VSMCs by aldosterone in all microarray, reverse transcriptase-polymerase chain reaction, immunoblotting, and immunofluorescence analyses. In addition, an analysis using small interfering RNA demonstrated that this gene product was involved in cell proliferation of VSMCs induced by aldosterone. Eplerenone, a specific MR antagonist, inhibited this gene induction by aldosterone in VSMCs. MDM2 protein was also more abundant in VSMCs of small resistance arteries in patients with primary aldosteronism compared with a control population. MDM2 is therefore considered one of the mineralocorticoid-responsive genes that regulates cell proliferation of VSMCs induced by MR-mediated aldosterone stimulation, possibly playing an important role in aldosterone-induced vascular structural remodeling.

Preparation and use of Coppersensor-1, a synthetic fluorophore for live-cell copper imaging

Coppersensor-1 (CS1) is a small-molecule, membrane-permeable fluorescent dye for imaging labile copper pools in biological samples, including live cells. This probe, comprising a boron dipyrromethene (BODIPY) chromophore coupled to a thioether-rich receptor, has a picomolar affinity for Cu+ with high selectivity over competing cellular metal ions. CS1 fluorescence increases up to 10-fold on binding to Cu+. In this protocol we describe the synthesis of CS1 and how to use this chemical tool to investigate intracellular levels of labile copper in cultured cells. The preparation of CS1 is anticipated to take 4-5 d, and imaging assays can be performed in 1-2 d with cultured cells.

Copper-dependent Interaction of Dynactin Subunit p62 with the N Terminus of ATP7B but Not ATP7A

The P-type ATPase affected in Wilson disease, ATP7B, is a key liver protein required to regulate and maintain copper homeostasis. When hepatocytes are exposed to elevated copper levels, ATP7B traffics from the trans-Golgi network toward the biliary canalicular membrane to excrete excess copper into bile. The N-terminal region of ATP7B contains six metal-binding sites (MBS), each with the copper-binding motif MXCXXC. These sites are required for the activity and copper-regulated intracellular redistribution of ATP7B. Two proteins are known to interact with the ATP7B N-terminal region: the copper chaperone ATOX1 that delivers copper to ATP7B, and COMMD1 (MURR1) that is potentially involved in vesicular copper sequestration. To identify additional proteins that interact with ATP7B and hence are involved in copper homeostasis, a yeast two-hybrid approach was employed to screen a human liver cDNA library. The dynactin subunit p62 (dynactin 4; DCTN4) was identified as an interacting partner, and this interaction was confirmed by co-immunoprecipitation from mammalian cells. The dynactin complex binds cargo, such as vesicles and organelles, to cytoplasmic dynein for retrograde microtubule-mediated trafficking and could feasibly be involved in the copper-regulated trafficking of ATP7B. The ATP7B/p62 interaction required copper, the metal-binding CXXC motifs, and the region between MBS 4 and MBS 6 of ATP7B. The p62 subunit did not interact with the related copper ATPase, ATP7A. We propose that the ATP7B interaction with p62 is a key component of the copper-induced trafficking pathway that delivers ATP7B to subapical vesicles of hepatocytes for the removal of excess copper into bile.

ATP7B Mediates Vesicular Sequestration of Copper: Insight Into Biliary Copper Excretion

The Wilson protein (ATP7B) regulates levels of systemic copper by excreting excess copper into bile. It is not clear whether ATP7B translocates excess intrahepatic copper directly across the canalicular membrane or sequesters this copper into exocytic vesicles, which subsequently fuse with canalicular membrane to expel their contents into bile. The aim of this study was to clarify the mechanism underlying ATP7B-mediated copper detoxification by investigating endogenous ATP7B localization in the HepG2 hepatoma cell line and its ability to mediate vesicular sequestration of excess intracellular copper. Immunofluorescence microscopy was used to investigate the effect of copper concentration on the localization of endogenous ATP7B in HepG2 cells. Copper accumulation studies to determine whether ATP7B can mediate vesicular sequestration of excess intracellular copper were performed using Chinese hamster ovary cells that exogenously expressed wild-type and mutant ATP7B proteins. In HepG2 cells, elevated copper levels stimulated trafficking of ATP7B to pericanalicular vesicles and not to the canalicular membrane as previously reported. Mutation of an endocytic retrieval signal in ATP7B caused the protein to constitutively localize to vesicles and not to the plasma membrane, suggesting that a vesicular compartment(s) is the final trafficking destination for ATP7B. Expression of wild-type and mutant ATP7B caused Chinese hamster ovary cells to accumulate copper in vesicles, which subsequently undergo exocytosis, releasing copper across the plasma membrane. This report provides compelling evidence that the primary mechanism of biliary copper excretion involves ATP7B-mediated vesicular sequestration of copper rather than direct copper translocation across the canalicular membrane.

Copper Induces Cytoplasmic Retention of Fission Yeast Transcription Factor Cuf1

Copper homeostasis within the cell is established and preserved by different mechanisms. Changes in gene expression constitute a way of maintaining this homeostasis. In Schizosaccharomyces pombe, the Cuf1 transcription factor is critical for the activation of copper transport gene expression under conditions of copper starvation. However, in the presence of elevated intracellular levels of copper, the mechanism of Cuf1 inactivation to turn off gene expression remains unclear. In this study, we provide evidence that inactivation of copper transport gene expression by Cuf1 is achieved through a copper-dependent, cytosolic retention of Cuf1. We identify a minimal nuclear localization sequence (NLS) between amino acids 11 to 53 within the Cuf1 N terminus. Deletion of this region and specific mutation of the Lys13, Arg16, Arg19, Lys24, Arg28, Lys45, Arg47, Arg50, and Arg53 residues to alanine within this putative NLS is sufficient to abrogate nuclear targeting of Cuf1. Under conditions of copper starvation, Cuf1 resides in the nucleus. However, in the presence of excess copper as well as silver ions, Cuf1 is sequestered in the cytoplasm, a process which requires the putative copper binding motif, 328Cys-X-Cys-X3-Cys-X-Cys-X2-Cys-X2-His342 (designated C-rich), within the C-terminal region of Cuf1. Deletion of this region and mutation of the Cys residues within the C-rich motif result in constitutive nuclear localization of Cuf1. By coexpressing the Cuf1 N terminus with its C terminus in trans and by using a two-hybrid assay, we show that these domains physically interact with each other in a copper-dependent manner. We propose a model wherein copper induces conformational changes in Cuf1 that promote a physical interaction between the Cuf1 N terminus and the C-rich motif in the C terminus that masks the NLS. Cuf1 is thereby sequestered in the cytosol under conditions of copper excess, thereby extinguishing copper transport gene expression.

Cop-like operon: Structure and organization in species of the Lactobacillale order

Copper is an essential and toxic trace metal for bacteria and, therefore, must be tightly regulated in the cell. Enterococcus hirae is a broadly studied model for copper homeostasis. The intracellular copper levels in E. hirae are regulated by the cop operon, which is formed by four genes: copA and copB that encode ATPases for influx and efflux of copper, respectively; copZ that encodes a copper chaperone; and copY, a copper responsive repressor. Since the complete genome sequence for E. hirae is not available, it is possible that other genes may encode proteins involved in copper homeostasis. Here, we identified a cop-like operon in nine species of Lactobacillale order with a known genome sequence. All of them always encoded a CopY-like repressor and a copper ATPase. The alignment of the cop-like operon promoter region revealed two CopY binding sites, one of which was conserved in all strains, and the second was only present in species of Streptococcus genus and L. johnsonii. Additional proteins associated to copper metabolism, CutC and Cupredoxin, also were detected. This study allowed for the description of the structure and organization of the cop operon and discussion of a phylogenetic hypothesis based on the differences observed in this operon's organization and its regulation in Lactobacillale order.

Gene expression profiling in wild-type and metallothionein mutant fibroblast cell lines

The role of metallothioneins (MT) in copper homeostasis is of great interest, as it appears to be partially responsible for the regulation of intracellular copper levels during adaptation to extracellular excess of the metal. To further investigate a possible role of MTs in copper metabolism, a genomics approach was utilized to evaluate the role of MT on gene expression. Microarray analysis was used to examine the effects of copper overload in fibroblast cells from normal and MT I and II double knock-out mice (MT-/-). As a first step, we compared genes that were significantly upregulated in wild-type and MT-/- cells exposed to copper. Even though wild-type and mutant cells are undistinguishable in terms of their morphological features and rates of growth, our results show that MT-/- cells do not respond with induction of typical markers of cellular stress under copper excess conditions, as observed in the wild-type cell line, suggesting that the transcription initiation rate or the mRNA stability of stress genes is affected when there is an alteration in the copper store capacity. The functional classification of other up-regulated genes in both cell lines indicates that a large proportion (>80%) belong to two major categories: 1) metabolism; and 2) cellular physiological processes, suggesting that at the transcriptional level copper overload induces the expression of genes associated with diverse molecular functions. These results open the possibility to understand how copper homeostasis is being coordinated with other metabolic pathways.

The Biosynthesis of Heme O and Heme A Is Not Regulated by Copper

Heme A is an obligatory cofactor in all eukaryotic and many prokaryotic cytochrome c oxidase (CcO) enzymes. Despite its obvious importance to CcO and the electron transport pathway, essentially nothing is known concerning the regulation of heme A. Because CcO is the only natural target for heme A and copper is also required for CcO activity, it was postulated that copper might regulate heme A homeostasis. Work reported previously demonstrated that there is often a strong connection between copper and iron homeostasis in general, and circumstantial evidence pointed to a possible specific link between copper and heme A. To address this question, we conducted experiments to determine rigorously whether copper plays a role in heme A homeostasis. The two enzymes responsible for the conversion of heme B to heme A, heme O synthase (HOS) and heme A synthase (HAS), were separately genomically epitope-tagged in Saccharomyces cerevisiae, and their expression under various copper conditions was quantified by Western blot analysis. These results demonstrated that the sum of transcription, translation, and stability of HOS and HAS were independent of copper. Additionally, the effects of intracellular copper concentrations on the activity of HOS and HAS from Bacillus subtilis (expressed in Escherichia coli) and Rhodobacter sphaeroides were examined by analysis of cellular heme extracts. No trends with respect to intracellular copper were observed. In combination, our results demonstrate that intracellular copper levels do not affect the transcription, translation, stability, or activity of either HOS or HAS.

Clioquinol Mediates Copper Uptake and Counteracts Copper Efflux Activities of the Amyloid Precursor Protein of Alzheimer's Disease

The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Abeta amyloid peptide. Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, knock-out animals revealed increased copper levels. A provoked rise in peripheral levels of copper reduced concentrations of soluble amyloid peptides and resulted in fewer pathogenic Abeta plaques. Contradictory evidence has been provided by the efficacy of copper chelation treatment with the drug clioquinol. Using a yeast model system, we show that adding clioquinol to the yeast culture medium drastically increased the intracellular copper concentration but there was no significant effect observed on zinc levels. This finding suggests that clioquinol can act therapeutically by changing the distribution of copper or facilitating copper uptake rather than by decreasing copper levels. The overexpression of the human APP or APLP2 extracellular domains but not the extracellular domain of APLP1 decreased intracellular copper levels. The expression of a mutant APP deficient for copper binding increased intracellular copper levels several-fold. These data uncover a novel biological function for APP and APLP2 in copper efflux and provide a new conceptual framework for the formerly diverging theories of copper supplementation and chelation in the treatment of Alzheimer's disease.

Intracellular trafficking of the human Wilson protein: the role of the six N-terminal metal-binding sites.

The Wilson protein (ATP7B) is a copper-transporting CPx-type ATPase defective in the copper toxicity disorder Wilson disease. In hepatocytes, ATP7B delivers copper to apo-ceruloplasmin and mediates the excretion of excess copper into bile. These distinct functions require the protein to localize at two different subcellular compartments. At the trans-Golgi network, ATP7B transports copper for incorporation into apo-ceruloplasmin. When intracellular copper levels are increased, ATP7B traffics to post-Golgi vesicles in close proximity to the canalicular membrane to facilitate biliary copper excretion. In the present study, we investigated the role of the six N-terminal MBSs (metal-binding sites) in the trafficking process. Using site-directed mutagenesis, we mutated or deleted various combinations of the MBSs and assessed the effect of these changes on the localization and trafficking of ATP7B. Results show that the MBSs required for trafficking are the same as those previously found essential for the copper transport function. Either MBS 5 or MBS 6 alone was sufficient to support the redistribution of ATP7B to vesicular compartments. The first three N-terminal motifs were not required for copper-dependent intracellular trafficking and could not functionally replace sites 4-6 when placed in the same sequence position. Furthermore, the N-terminal region encompassing MBSs 1-5 (amino acids 64-540) was not essential for trafficking, with only one MBS close to the membrane channel, necessary and sufficient to support trafficking. Our findings were similar to those obtained for the closely related ATP7A protein, suggesting similar mechanisms for trafficking between copper-transporting CPx-type ATPases.

A novel role for XIAP in copper homeostasis through regulation of MURR1.

XIAP is a potent suppressor of apoptosis that directly inhibits specific members of the caspase family of cysteine proteases. Here we demonstrate a novel role for XIAP in the control of intracellular copper levels. XIAP was found to interact with MURR1, a factor recently implicated in copper homeostasis. XIAP binds to MURR1 in a manner that is distinct from that utilized by XIAP to bind caspases, and consistent with this, MURR1 did not affect the antiapoptotic properties of XIAP. However, cells and tissues derived from Xiap-deficient mice were found to contain reduced copper levels, while suppression of MURR1 resulted in increased intracellular copper in cultured cells. Consistent with these opposing effects, XIAP was observed to negatively regulate MURR1 protein levels by the formation of K48 polyubiquitin chains on MURR1 that promote its degradation. These findings represent the first described phenotypic alteration in Xiap-deficient mice and demonstrate that XIAP can function through MURR1 to regulate copper homeostasis.

Influence of zinc and copper ions on metals transport, antioxidant system responses and membrane LPO levels of F. equiseti and F. acuminatum

This study has highlighted the role of copper and zinc ions on the intracellular metal levels as well as antioxidant enzyme activities and membrane lipid peroxidation levels in Fusarium equiseti and Fusarium acuminatum. Elevating zinc concentration in the growth medium from 2 to 6 μM Zn 2+ results the increases in intracellular zinc levels and the decreases in iron and manganese levels. However, magnesium levels have been unaltered depending on the Zn 2+ concentration. On the other hand, SOD and CAT activities of F. equiseti and F. acuminatum grown in 4 μM Zn 2+ were reached the maximum values where the intracellular manganese and iron levels have been determined to be optimum values. These results suggest that the metals, which are cofactor of these enzymes, are effective elicitors. In addition, intracellular copper, zinc and iron levels have been indicated positive correlation with copper concentration of the growth medium. In contrast, manganese levels have been decreased and magnesium levels unaltered with respect to increasing of copper concentration. In the presence of copper, significant inhibition of antioxidant enzyme was observed, while the increases in membrane lipid peroxidation levels were detected in the cells. The results may suggest that the enzymes were inactivated by free radicals produced in Haber–Weiss reaction, which was initiated by toxic concentration of copper and iron metals.

Severe reduction of superoxide dismutase activity in the yeast Saccharomyces cerevisae with the deletion or overexpression of GTS1

We report herein that the level of reactive oxygen species (ROS) observed using dihydrorhodamine is much higher in either GTS1-deleted ( gts1Δ) or GTS1-overexpressing (TMp GTS1) transformants than in the wild-type and that the levels of protein carbonyls are increased and the glutathione levels are decreased in both transformants. Consistently, the activities of superoxide dismutases (SODs) in both gts1Δ and TMp GTS1 were severely weakened, while the protein levels of both Cu/Zn-SOD and Mn-SOD were not so changed. As the intracellular copper levels were significantly increased in both transformants, we hypothesized that, in either gts1Δ or TMp GTS1 cells, the imbalanced homeostasis of copper induced an accumulation of ROS which caused inactivation of SODs further increasing ROS levels.

The metallochaperone Atox1 plays a critical role in perinatal copper homeostasis.

Copper plays a fundamental role in the biochemistry of all aerobic organisms. The delivery of this metal to specific intracellular targets is mediated by metallochaperones. To elucidate the role of the metallochaperone Atox1, we analyzed mice with a disruption of the Atox1 locus. Atox1(-/-) mice failed to thrive immediately after birth, with 45% of pups dying before weaning. Surviving animals exhibited growth failure, skin laxity, hypopigmentation, and seizures because of perinatal copper deficiency. Maternal Atox1 deficiency markedly increased the severity of Atox1(-/-) phenotype, resulting in increased perinatal mortality as well as severe growth retardation and congenital malformations among surviving Atox1(-/-) progeny. Furthermore, Atox1-deficient cells accumulated high levels of intracellular copper, and metabolic studies indicated that this defect was because of impaired cellular copper efflux. Taken together, these data reveal a direct role for Atox1 in trafficking of intracellular copper to the secretory pathway of mammalian cells and demonstrate that this metallochaperone plays a critical role in perinatal copper homeostasis.

Pyrrolidine Dithiocarbamate Inhibits TNF-α-Dependent Activation of NF-κB by Increasing Intracellular Copper Level in Human Aortic Smooth Muscle Cells

Pyrrolidine dithiocarbamate (PDTC) is a metal-chelating compound that acts as antioxidant or pro-oxidant and is widely used to study redox regulation of cell function. In the present study, we investigated effects of PDTC and another antioxidant, N-acetyl-l-cysteine (NAC), on TNF-α-dependent activation of NF-κB in human aortic smooth muscle cells (HASMC). Treatment of the cells with TNF-α induced the activation of p65/p50 heterodimer NF-κB and increased the mRNA levels of monocyte chemoattractant protein (MCP)-1. Pretreatment with PDTC markedly suppressed the NF-κB activation and expression of MCP-1 by inhibiting IκB-α degradation. In contrast, NAC had no effect. PDTC concomitantly increased the intracellular levels of copper, and bathocuproinedisulfonic acid, a non-cell-permeable chelator of Cu1+, inhibited the PDTC-induced increase in intracellular copper level and reversed the PDTC effects on IκB-α, NF-κB, and MCP-1. These results indicate that TNF-α-dependent expression of MCP-1 in HASMC is tightly regulated by NF-κB and that intracellular copper level is crucial for the TNF-α-dependent activation of NF-κB in HASMC.

Effect of zinc on vascular smooth muscle cell death mediated by PDTC

Pyrrolidinedithiocarbamate (PDTC) andN-Acetylcysteine (NAC) are metal and nonmetal-chelating antioxidant which can induce rat and human smooth muscle cell death. When the smooth muscle cells from mouse aorta (MASMC) that we successfully cultured recently was exposed to PDTC and NAC in a normal serum state, the cells were induced to death by these compounds. However, PDTC did not induce the cell death in a serum depleted medium. This data suggests that certain factors in the serum may mediate the cytotoxic effect of PDTC. The metal chelator, Ca-EDTA blocked PDTC-induced cell death, but Cu-, Fe-, and Zn-EDTA did not block the PDTC-induced cell death. This data indicated that copper, iron, and zinc in the serum may lead to the cytotoxic effect of PDTC Investigation of the intracellular zinc level in PDTC-induced smooth muscle cell death using the zinc probe dyeN-(6-methoxy-8-quinolyl)-p-toluenesulfonamide shows that only the musclecontaining layers of the arteries have higher level of zinc. As expected, PDTC increased the intracellular fluorescence level of the zinc. In agreement with these results, the addition of an exogenous metal, zinc, induced the vascular aortic smooth muscle cell death which led to an increased intracellular zinc level. We concluded that PDTC induced mouse aortic smooth muscle cell death required not only zinc level but also intracellular copper and iron level. The mechanism of this antioxidant to induce vascular smooth muscle cell death may provide a new strategy to prevent their proliferation in arteriosclerotic lesions.

Copper inhibits β-amyloid production and stimulates the non-amyloidogenic pathway of amyloid-precursor-protein secretion

Previous studies have demonstrated that amyloid precursor protein (APP) can bind and reduce Cu(II) to Cu(I), leading to oxidative modification of APP. In the present study we show that adding copper to Chinese-hamster ovary (CHO) cells greatly reduced the levels of amyloid Aβ peptide (Aβ) both in parental CHO-K1 and in copper-resistant CHO-CUR3 cells, which have lower intracellular copper levels. Copper also caused an increase in the secretion of the APP ectodomain, indicating that the large decrease in Aβ release was not due to a general inhibition in protein secretion. There was an increase in intracellular full-length APP levels which paralleled the decrease in Aβ generation, suggesting the existence of two distinct regulating mechanisms, one acting on Aβ production and the other on APP synthesis. Maximal inhibition of Aβ production and stimulation of APP secretion was achieved in CHO-K1 cells at about 10 μM copper and in CHO-CUR3 cells at about 50 μM copper. This dose ‘window of opportunity’ at which copper promoted the non-amyloidogenic pathway of APP was confirmed by an increase in the non-amyloidogenic p3 fragment produced by α-secretase cleavage. Our findings suggest that copper or copper agonists might be useful tools to discover novel targets for anti-Alzheimer drugs and may prove beneficial for the prevention of Alzheimer's disease.

Copper inhibits β-amyloid production and stimulates the non-amyloidogenic pathway of amyloid-precursor-protein secretion

Previous studies have demonstrated that amyloid precursor protein (APP) can bind and reduce Cu(II) to Cu(I), leading to oxidative modification of APP. In the present study we show that adding copper to Chinese-hamster ovary (CHO) cells greatly reduced the levels of amyloid Abeta peptide (Abeta) both in parental CHO-K1 and in copper-resistant CHO-CUR3 cells, which have lower intracellular copper levels. Copper also caused an increase in the secretion of the APP ectodomain, indicating that the large decrease in Abeta release was not due to a general inhibition in protein secretion. There was an increase in intracellular full-length APP levels which paralleled the decrease in Abeta generation, suggesting the existence of two distinct regulating mechanisms, one acting on Abeta production and the other on APP synthesis. Maximal inhibition of Abeta production and stimulation of APP secretion was achieved in CHO-K1 cells at about 10 microM copper and in CHO-CUR3 cells at about 50 microM copper. This dose 'window of opportunity' at which copper promoted the non-amyloidogenic pathway of APP was confirmed by an increase in the non-amyloidogenic p3 fragment produced by alpha-secretase cleavage. Our findings suggest that copper or copper agonists might be useful tools to discover novel targets for anti-Alzheimer drugs and may prove beneficial for the prevention of Alzheimer's disease.