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 Aβ 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 Aβ 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. The key protein in Alzheimer's disease, the amyloid precursor protein (APP), is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Aβ 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 Aβ 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. The amyloid precursor protein (APP) 1The abbreviations used are: APP, amyloid precursor protein; Aβ, amyloid β; CuBD, copper-binding domain; AD, Alzheimer's disease; CQ, clioquinol; sAPP, secreted APP; APLP, amyloid precursor-like protein; sAPLP, secreted APLP; ICP-MS, inductively coupled mass spectrometry; SOD, superoxide dismutase; ySOD, yeast SOD. of Alzheimer's disease (AD) is a ubiquitously expressed copper-binding glycoprotein that gives rise to the Aβ amyloid peptide (1Selkoe D.J. Schenk D. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 545-584Crossref PubMed Scopus (745) Google Scholar). The APP paralogs and orthologs demonstrate a significant evolutionary change in the function of the N-terminal copper-binding domain (CuBD). In higher species, CuBD has a gain-in activity toward promoting Cu(II) reduction (2Simons A. Ruppert T. Schmidt C. Schlicksupp A. Pipkorn R. Reed J. Masters C.L. White A.R. Cappai R. Beyreuther K. Bayer T.A. Multhaup G. Biochemistry. 2002; 41: 9310-9320Crossref PubMed Scopus (46) Google Scholar, 3White A.R. Multhaup G. Galatis D. McKinstry W.J. Parker M.W. Pipkorn R. Beyreuther K. Masters C.L. Cappai R. J. Neurosci. 2002; 22: 365-376Crossref PubMed Google Scholar). Copper deprivation strongly down-regulates APP gene expression and reduces APP levels (4Bellingham S.A. Lahiri D.K. Maloney B. La Fontaine S. Multhaup G. Camakaris J. J. Biol. Chem. 2004; 279: 20378-20386Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Animal model systems revealed that APP is actively involved in balancing copper concentrations in vivo. Whereas overexpression of APP results in significantly reduced brain copper levels in three different lines of transgenic mice, APP23, Tg2576, and TgCRND8 (5Bayer T.A. Schafer S. Simons A. Kemmling A. Kamer T. Tepest R. Eckert A. Schussel K. Eikenberg O. Sturchler-Pierrat C. Abramowski D. Staufenbiel M. Multhaup G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14187-14192Crossref PubMed Scopus (310) Google Scholar, 6Maynard C.J. Cappai R. Volitakis I. Cherny R.A. White A.R. Beyreuther K. Masters C.L. Bush A.I. Li Q.X. J. Biol. Chem. 2002; 277: 44670-44676Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 7Phinney A.L. Drisaldi B. Schmidt S.D. Lugowski S. Coronado V. Liang Y. Horne P. Yang J. Sekoulidis J. Coomaraswamy J. Chishti M.A. Cox D.W. Mathews P.M. Nixon R.A. Carlson G.A. St George-Hyslop P. Westaway D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14193-14198Crossref PubMed Scopus (207) Google Scholar), conversely, APP and APLP2 knock-out mouse revealed increased copper levels in cerebral cortex and liver (8White A.R. Reyes R. Mercer J.F. Camakaris J. Zheng H. Bush A.I. Multhaup G. Beyreuther K. Masters C.L. Cappai R. Brain Res. 1999; 842: 439-444Crossref PubMed Scopus (259) Google Scholar). Whereas the relevance of a disturbed metal ion homeostasis in Alzheimer's disease is presently unclear (9Squitti R. Lupoi D. Pasqualetti P. Dal Forno G. Vernieri F. Chiovenda P. Rossi L. Cortesi M. Cassetta E. Rossini P.M. Neurology. 2002; 59: 1153-1161Crossref PubMed Scopus (210) Google Scholar, 10Samudralwar D.L. Diprete C.C. Ni B.F. Ehmann W.D. Markesbery W.R. J. Neurol. Sci. 1995; 130: 139-145Abstract Full Text PDF PubMed Scopus (110) Google Scholar, 11Thompson C.M. Markesbery W.R. Ehmann W.D. Mao Y.X. Vance D.E. Neurotoxicology. 1988; 9: 1-7PubMed Google Scholar), studies in two different transgenic mouse models convincingly demonstrate that a rise in peripheral copper levels reduced soluble Aβ peptide levels (5Bayer T.A. Schafer S. Simons A. Kemmling A. Kamer T. Tepest R. Eckert A. Schussel K. Eikenberg O. Sturchler-Pierrat C. Abramowski D. Staufenbiel M. Multhaup G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14187-14192Crossref PubMed Scopus (310) Google Scholar) and the number of Aβ plaques (7Phinney A.L. Drisaldi B. Schmidt S.D. Lugowski S. Coronado V. Liang Y. Horne P. Yang J. Sekoulidis J. Coomaraswamy J. Chishti M.A. Cox D.W. Mathews P.M. Nixon R.A. Carlson G.A. St George-Hyslop P. Westaway D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14193-14198Crossref PubMed Scopus (207) Google Scholar). In a mammalian cell line, increasing intracellular copper levels attenuated APP processing into Aβ and stimulated secreted APP levels (5Bayer T.A. Schafer S. Simons A. Kemmling A. Kamer T. Tepest R. Eckert A. Schussel K. Eikenberg O. Sturchler-Pierrat C. Abramowski D. Staufenbiel M. Multhaup G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14187-14192Crossref PubMed Scopus (310) Google Scholar, 12Borchardt T. Camakaris J. Cappai R. Masters C.L. Beyreuther K. Multhaup G. Biochem. J. 1999; 344: 461-467Crossref PubMed Scopus (159) Google Scholar). This finding demonstrates that copper-binding to APP alters the turnover of brain APP and concomitantly offers an alternative therapeutic intervention. Based on earlier observations that copper and chelators have an effect on amyloid fibril formation (13Cherny R.A. Legg J.T. McLean C.A. Fairlie D.P. Huang X. Atwood C.S. Beyreuther K. Tanzi R.E. Masters C.L. Bush A.I. J. Biol. Chem. 1999; 274: 23223-23228Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar), the Cu,Zn chelator clioquinol was administered to APP-overexpressing Tg2576 mice (14Cherny R.A. Atwood C.S. Xilinas M.E. Gray D.N. Jones W.D. McLean C.A. Barnham K.J. Volitakis I. Fraser F.W. Kim Y. Huang X. Goldstein L.E. Moir R.D. Lim J.T. Beyreuther K. Zheng H. Tanzi R.E. Masters C.L. Bush A.I. Neuron. 2001; 30: 665-676Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar). Intriguingly, the oral treatment with clioquinol (CQ), despite being a chelator for copper and zinc, significantly elevated brain copper by 19% and zinc by 13%. It also elevated the concentration of soluble brain Aβ by 50%, whereas Aβ deposition was markedly inhibited (14Cherny R.A. Atwood C.S. Xilinas M.E. Gray D.N. Jones W.D. McLean C.A. Barnham K.J. Volitakis I. Fraser F.W. Kim Y. Huang X. Goldstein L.E. Moir R.D. Lim J.T. Beyreuther K. Zheng H. Tanzi R.E. Masters C.L. Bush A.I. Neuron. 2001; 30: 665-676Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar). These results lead to the conclusion that CQ did not induce a loss in metal ions systematically. Possibly, CQ might have redistributed copper from plaques to the cells, or alternatively, facilitated the uptake of CQ-copper complexes in the brain because CQ is hydrophobic and crosses the blood-brain-barrier (BBB). In a Phase II clinical trial, CQ inhibited cognitive decline and decreased plasma Aβ42 levels in moderate to severe AD patients (15Ritchie C.W. Bush A.I. Mackinnon A. Macfarlane S. Mastwyk M. MacGregor L. Kiers L. Cherny R. Li Q.X. Tammer A. Carrington D. Mavros C. Volitakis I. Xilinas M. Ames D. Davis S. Beyreuther K. Tanzi R.E. Masters C.L. Arch. Neurol. 2003; 60: 1685-1691Crossref PubMed Scopus (901) Google Scholar). Taken together, these findings jointly argue that APP and its paralogs may play a critical role in intracellular copper homeostasis and we anticipate that (1Selkoe D.J. Schenk D. Annu. Rev. Pharmacol. Toxicol. 2003; 43: 545-584Crossref PubMed Scopus (745) Google Scholar) APP or APLP2 expression is a substantial driving force for copper efflux and (2Simons A. Ruppert T. Schmidt C. Schlicksupp A. Pipkorn R. Reed J. Masters C.L. White A.R. Cappai R. Beyreuther K. Bayer T.A. Multhaup G. Biochemistry. 2002; 41: 9310-9320Crossref PubMed Scopus (46) Google Scholar) CQ treatment enables copper passage into the cell. To test these hypotheses, the methylotrophic yeast Pichia pastoris was used to examine the effect of expression and secretion of the N-terminal domains of human APP (sAPP), APLP1 (sAPLP1), or APLP2 (sAPLP2) on intracellular concentrations of copper in yeast cells and the influence of CQ on the APP-expressing cells (16Henry A. Masters C.L. Beyreuther K. Cappai R. Protein Expression Purif. 1997; 10: 283-291Crossref PubMed Scopus (49) Google Scholar). Our studies showed that clioquinol in yeast culture medium drastically increased the intracellular copper concentration in wild-type and APP-expressing cells. These results indicated that the reported protective effects of CQ are not considered solely a chelator of zinc and/or copper. In addition, we reported that APP and APLPL2 but not APLP1 are involved in copper homeostasis as copper efflux proteins. Expression Constructs and Site-directed Mutagenesis—The APP18–350 (sAPP) construct in pPIZαA (Invitrogen) was generated by PCR using the EcoRI and NotI sites in a 5′-overhang region of the forward primer (APP18–350fwd, 5′-GC GGG GCC GAA TTC CTG GAG GTA CCC ACT GAT GGT AAT G-3′) and the reverse primer (APP18–350rev, 5′-TTC ATC TAA GCG GCC GCT TAG AGT AAA CTT TGG GAC ATG GCG CTG CCA CAC AC-3′), respectively. Site-directed mutagenesis of APP18–350 mutant was performed with PCR using a mismatch forward primer (APP18–350AAAfwd, 5′-AGG AGA GGA TGG ATG TTT GCG AAA CTG CTC TTG CCT GGG CCA CCG TCG CCA AAG AGA C-3′) and the The generated was used with the primer on the The into the EcoRI and NotI sites to the The with of P. pastoris by cells showed the secreted APLP2 (sAPLP2) and APLP1 in P. pastoris by using the (Invitrogen) as has been A.R. Zheng H. Galatis D. F. L. Multhaup G. Beyreuther K. Masters C.L. Cappai R. J. Neurosci. PubMed Google Scholar). Expression in P. of was on yeast for a test used to medium yeast yeast and to an of cells and to an of in medium with of to induce and for of different to the for and to expression levels by in yeast in to an of and in and and cell for protein of cell was performed by adding of and the of by of for and for on the cell and the of the culture with for and on a to by for binding sites with with the in and with the The of was performed by using the to the by the and of the using a for in of medium with or or and the cell was of yeast culture in in in in in and in for the with and an of metal by and in to a concentration of for by was performed by using a in with mass three three and an of as (2Simons A. Ruppert T. Schmidt C. Schlicksupp A. Pipkorn R. Reed J. Masters C.L. White A.R. Cappai R. Beyreuther K. Bayer T.A. Multhaup G. Biochemistry. 2002; 41: 9310-9320Crossref PubMed Scopus (46) Google Scholar). 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A. 2003; 100: 14193-14198Crossref PubMed Scopus (207) Google Scholar), the expression of decreased the intracellular copper in yeast cells by with cells as by was a but not significant in zinc levels cells and cells and the cells in the of the concentration of copper in the cells significantly (2Simons A. Ruppert T. Schmidt C. Schlicksupp A. Pipkorn R. Reed J. Masters C.L. White A.R. Cappai R. Beyreuther K. Bayer T.A. Multhaup G. Biochemistry. 2002; 41: 9310-9320Crossref PubMed Scopus (46) Google Scholar, T.A. Schafer S. Simons A. Kemmling A. Kamer T. Tepest R. Eckert A. Schussel K. Eikenberg O. Sturchler-Pierrat C. Abramowski D. Staufenbiel M. Multhaup G. Proc. Natl. Acad. Sci. U. S. 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Galatis D. C.J. C.C. White A.R. Beyreuther K. Masters C.L. Parker M.W. Cappai R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) in APP a and and for its influence on intracellular copper levels. P. pastoris cells that copper supplementation the of clioquinol significantly the expression levels of wild-type or mutant as by the a and the copper levels of the cells showed a with cells. In the of intracellular copper levels are increased because of the expression of the mutant construct a and These results that the CuBD of is for the copper efflux activity of the copper concentrations in the medium the copper concentrations in the cells and the secretion of as revealed by an by This finding suggests that the expression of may with copper or distribution systems in yeast cells Schmidt R.A. 1999; PubMed Scopus Google Scholar, H. J. 2003; PubMed Google Scholar, J. Xilinas M. L. J. Neurol. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). To the reduced copper in or cells the of we the activity of in yeast cells in or in medium by using a D. L. D.L. J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). was a of activity in cells and a of activity in cells that not observed for with cells. The overexpression of mutant resulted in a in J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar) activity that with the intracellular copper levels the expression of or the intracellular copper for but not whereas mutant inhibited copper efflux and increased the intracellular of copper to To test the that CQ can the uptake of copper the formation of a CQ-copper cells with CQ or with of CQ and in the was a significant of cell in the of CQ or CQ and The effects of CQ by adding an of copper or This not with metal ions as or not CQ was for the not by of the In the was that CQ to and that was by a to a chelation of from by CQ J. Xilinas M. L. J. Neurol. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). was facilitated uptake of copper into yeast cells in medium with CQ copper. A of intracellular copper levels was in cells with CQ-copper with cells with copper In the of CQ, and the of intracellular copper was increased to This effect was for copper because the treatment did not change zinc levels In the of CQ, of the a effect on intracellular copper concentrations medium supplementation with a significant of zinc levels in cells an effect that was by the expression of sAPP, that is involved in the of the zinc This finding suggests that the by CQ was rather to a facilitated uptake of copper than to a as for the elevated copper and zinc levels in mice (14Cherny R.A. Atwood C.S. Xilinas M.E. Gray D.N. Jones W.D. McLean C.A. Barnham K.J. Volitakis I. Fraser F.W. Kim Y. Huang X. Goldstein L.E. Moir R.D. Lim J.T. Beyreuther K. Zheng H. Tanzi R.E. Masters C.L. Bush A.I. Neuron. 2001; 30: 665-676Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar). The role of CQ in copper uptake was in yeast expression to the of intracellular copper of the APP secretion the effect of CQ on the intracellular copper the intracellular copper in cells was to a with a in cells in the of CQ-copper in the a of in cells and of in cells was observed This attenuated an of copper levels in cells the observed efflux from expression rather than from The in results that or secretion can copper The of the copper-binding mutant to copper efflux that the copper the binding of copper to the CuBD of The of copper from concentrations to in the medium activity in cells and the number of copper cell increased by Whereas activity was by and but not by APLP1 expression or copper the expression of the mutant increased activity and the number of copper cell. In the of the intracellular copper increased by the activity was increased and the of copper the cell is a key to activity J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), copper have been to in mutant cells but not in cells. the overexpression of APP in three different Tg2576, APP23, and TgCRND8 mice, resulted in significantly reduced brain copper levels the of (5Bayer T.A. Schafer S. Simons A. Kemmling A. Kamer T. Tepest R. Eckert A. Schussel K. Eikenberg O. Sturchler-Pierrat C. Abramowski D. Staufenbiel M. Multhaup G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14187-14192Crossref PubMed Scopus (310) Google Scholar, 6Maynard C.J. Cappai R. Volitakis I. Cherny R.A. White A.R. Beyreuther K. Masters C.L. Bush A.I. Li Q.X. J. Biol. Chem. 2002; 277: 44670-44676Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar, 7Phinney A.L. Drisaldi B. Schmidt S.D. Lugowski S. Coronado V. Liang Y. Horne P. Yang J. Sekoulidis J. Coomaraswamy J. Chishti M.A. Cox D.W. Mathews P.M. Nixon R.A. Carlson G.A. St George-Hyslop P. Westaway D. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14193-14198Crossref PubMed Scopus (207) Google Scholar), the effects by an in copper homeostasis and intracellular copper The results provide a to and a role for secreted APP in efflux of copper. Based on the to copper that also the APP CuBD may function as a and/or K.J. McKinstry W.J. Multhaup G. Galatis D. C.J. C.C. White A.R. Beyreuther K. Masters C.L. Parker M.W. Cappai R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of domain and the and of and are involved in copper a novel copper-binding is K.J. McKinstry W.J. Multhaup G. Galatis D. C.J. C.C. White A.R. Beyreuther K. Masters C.L. Parker M.W. Cappai R. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The of the APP from the secreted to copper or a copper in P. The the effect of mutant with the but in a and copper levels. The that the reduction of intracellular copper is an of a chelator effect by or in the medium is because with a of of copper. The data with and a that copper binding to the CuBD of APP G. Schlicksupp A. L. D. Ruppert T. Masters C.L. Beyreuther K. PubMed Scopus Google Scholar) and to Aβ C.S. Moir R.D. Huang X. M.A. Tanzi R.E. Bush A.I. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) is an It was that was by a to the chelation of by CQ J. Xilinas M. L. J. Neurol. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar). Our data the that CQ can to the by a with but in the of the metal ions copper or increased zinc levels in cells. observations indicated that levels and zinc are in the and that to the was to on the of zinc C.J. J. Google Scholar, J. 2003; PubMed Scopus Google Scholar). A may in P. the of model to metal ion by being a CQ did not induce a loss in levels but rather inhibited formation and concomitantly increased soluble brain copper and zinc levels (14Cherny R.A. Atwood C.S. Xilinas M.E. Gray D.N. Jones W.D. McLean C.A. Barnham K.J. Volitakis I. Fraser F.W. Kim Y. Huang X. Goldstein L.E. Moir R.D. Lim J.T. Beyreuther K. Zheng H. Tanzi R.E. Masters C.L. Bush A.I. Neuron. 2001; 30: 665-676Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar). CQ facilitated the uptake of copper as CQ-copper to mice C. M. Res. Chem. Pharmacol. Google Scholar). In CQ-copper complexes in the and the to the brain and soluble copper and zinc levels increased by CQ treatment in mice brain (14Cherny R.A. Atwood C.S. Xilinas M.E. Gray D.N. Jones W.D. McLean C.A. Barnham K.J. Volitakis I. Fraser F.W. Kim Y. Huang X. Goldstein L.E. Moir R.D. Lim J.T. Beyreuther K. Zheng H. Tanzi R.E. Masters C.L. Bush A.I. Neuron. 2001; 30: 665-676Abstract Full Text Full Text PDF PubMed Scopus (1322) Google Scholar). CQ-copper complexes and markedly copper levels in the brain of with AD and the in copper levels observed in AD, the APP the that APP transgenic mice with copper than mice (5Bayer T.A. Schafer S. Simons A. Kemmling A. Kamer T. Tepest R. Eckert A. Schussel K. Eikenberg O. Sturchler-Pierrat C. Abramowski D. Staufenbiel M. Multhaup G. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 14187-14192Crossref PubMed Scopus (310) Google Scholar), these may in the of copper homeostasis in AD and with as for and G. T. and M. for