Degradation of the Alzheimer Disease Amyloid β-Peptide by Metal-dependent Up-regulation of Metalloprotease Activity (2006)

Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid β-peptide (Aβ) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu2+ or Zn2+ resulted in an ∼85–90% reduction of secreted Aβ-(1–40) and Aβ-(1–42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted Aβ was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu2+. MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu2+ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of Aβ-(1–40). Our findings identify an alternative mechanism of action for CQ in the reduction of Aβ deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients. Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid β-peptide (Aβ) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu2+ or Zn2+ resulted in an ∼85–90% reduction of secreted Aβ-(1–40) and Aβ-(1–42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted Aβ was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu2+. MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu2+ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of Aβ-(1–40). Our findings identify an alternative mechanism of action for CQ in the reduction of Aβ deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients. Alzheimer disease (AD) 4The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β-peptide; APP, amyloid precursor protein; MMPs, matrix metalloproteases; CQ, clioquinol; CHO, Chinese hamster ovary; BPS, bathophenanthroline disulfonate; GSK, glycogen synthase kinase; MnTMPyP, Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; ICP-MS, inductively coupled plasma mass spectrometry; PI3K, phosphoinositol 3-kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MAPK, mitogen-activated protein kinase. 4The abbreviations used are: AD, Alzheimer disease; Aβ, amyloid β-peptide; APP, amyloid precursor protein; MMPs, matrix metalloproteases; CQ, clioquinol; CHO, Chinese hamster ovary; BPS, bathophenanthroline disulfonate; GSK, glycogen synthase kinase; MnTMPyP, Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin pentachloride; JNK, c-Jun N-terminal kinase; ERK, extracellular signal-regulated kinase; ICP-MS, inductively coupled plasma mass spectrometry; PI3K, phosphoinositol 3-kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; ELISA, enzyme-linked immunosorbent assay; PBS, phosphate-buffered saline; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; MAPK, mitogen-activated protein kinase. is characterized by progressive neuronal dysfunction, reactive gliosis, and the formation of amyloid plaques in the brain. The major constituent of AD plaques is the amyloid β-peptide (Aβ), which is cleaved from the membrane-bound amyloid precursor protein (APP) (1Masters C.L. Simms G. Weinman N.A. Multhaup G. McDonald B.L. Beyreuther K. Proc. Natl. Acad. Sci. U. S. A. 1985; 82: 4245-4249Crossref PubMed Scopus (3660) Google Scholar). Aggregated or oligomeric Aβ can induce neurotoxicity through pathways involving free radical production and increased neuronal oxidative stress (2Barnham K.J. Masters C.L. Bush A.I. Nat. Rev. Drug Discov. 2004; 3: 205-214Crossref PubMed Scopus (2751) Google Scholar). Among the factors capable of promoting Aβ aggregation in vivo, recent evidence supports a central role for biometals such as Cu2+ and Zn2+ in this process (3Bush A.I. Trends Neurosci. 2003; 26: 207-214Abstract Full Text Full Text PDF PubMed Scopus (1127) Google Scholar). An important factor in controlling Aβ accumulation in AD patients is the activity of Aβ-degrading enzymes. Recent studies have identified several candidate proteases that may contribute to catabolism of Aβ in the brain. Neprilysin, insulin-degrading enzyme, angiotensin-converting enzyme, and matrix metalloproteases (MMPs) have all demonstrated Aβ-degrading activity in vitro and/or in vivo (4Roher A.E. Kasunic T.C. Woods A.S. Ball M.J. Fridman R. Biochem. Biophys. Res. Commun. 1994; 205: 1755-1761Crossref PubMed Scopus (124) Google Scholar, 5Howell S. Nalbantoglu J. Crine P. Peptides (N.Y.). 1995; 16: 647-652Crossref PubMed Scopus (186) Google Scholar, 6Backstrom J.R. Lim G.P. Cullen M.J. Tokes Z.A. J. Neurosci. 1996; 16: 7910-7919Crossref PubMed Google Scholar). Reduced activity of these or other Aβ-degrading proteases with age may play a role in promoting accumulation and deposition of Aβ in AD patients. Development of strategies to enhance clearance of Aβ may lead to novel therapeutic treatments for AD patients. Promoting Aβ clearance may be achieved through modulating metal sequestration or metal-protein interactions. 5-Chloro-7-iodo-8-hydroxyquinoline or clioquinol (CQ), a disused antibiotic, has received considerable attention as a potential metal ligand in AD and Parkinson disease patients (7Kaur D. Yantiri F. Rajagopalan S. Kumar J. Mo J.Q. Boonplueang R. Viswanath V. Jacobs R. Yang L. Beal M.F. DiMonte D. Volitakis I. Ellerby L. Cherny R.A. Bush A.I. Andersen J.K. Neuron. 2003; 37: 899-909Abstract Full Text Full Text PDF PubMed Scopus (556) Google Scholar, 8Ritchie 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 (900) Google Scholar, 9Ritchie C.W. Bush A.I. Masters C.L. Expert Opin. Investig. Drugs. 2004; 13: 1585-1592Crossref PubMed Scopus (56) Google Scholar). Preliminary studies revealed that CQ rapidly and potently dissolved aggregates of synthetic or AD brain-derived Aβ in vitro (10Cherny 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 (1321) Google Scholar). In subsequent animal studies, a 9-week oral treatment with CQ resulted in a 49% reduction of Aβ levels and significantly increased Cu2+ and Zn2+ levels in brains of Tg2576 mice (10Cherny 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 (1321) Google Scholar). Small clinical trials of CQ have demonstrated a significant slowing of cognitive decline together with a lowering of plasma Aβ-(1–42) levels in a subset of AD patients compared with matched placebo controls (8Ritchie 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 (900) Google Scholar). The mechanism of action by CQ was suggested to be via metal sequestration, resulting in Aβ CQ also by alternative pathways involving modulation of or Aβ C. A. M. M. R. Multhaup G. J. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). Chinese hamster ovary cells overexpressing were with CQ in the or of levels of CQ was to cells in the of Cu2+ or the secreted levels of Aβ-(1–40) and Aβ-(1–42) were Analogous effects were seen in neuroblastoma cells. revealed that this effect was with of Cu2+ and Zn2+ and loss of Aβ through increased findings identify a novel mechanism for the therapeutic of CQ in which or Aβ bathophenanthroline and were from glycogen synthase kinase MMP MMP MMP-3 Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin and were from was from was a from to or of JNK, and were from of and and neuroblastoma cells were by the in the were and in with and from cells were and of to CQ and cells were a of and in or for CQ was as a in and to with as described metal levels in the were and for and as by inductively coupled plasma mass were and the was by to addition to cells. were with of phosphoinositol 3-kinase and JNK and metalloproteases and were as in and the MnTMPyP, BPS, and MMP-3 were as in was as a in or cells were to synthetic Aβ-(1–40) with or CQ, and were for to and were for of Aβ levels by enzyme-linked immunosorbent was by cells were and cells were with phosphate-buffered and for of metal levels by were with CQ and/or for and with were an was for protein and the cells were by for in a levels were in cell by as described R. J. Neurosci. Res. 2003; PubMed Scopus Google and to of of of Aβ-(1–40) was from the M. and dissolved in The dissolved was addition to cell in In Aβ-(1–40) was also to or cells in or addition of and CQ, or CQ and the was and the Aβ-(1–40) levels were by for Aβ levels were in the were with in and for Aβ-(1–40) were with and with and the or Aβ were to and were with and was The were was and the were in a with and Aβ-(1–40) and Aβ-(1–42) and were in The from the were used to the Aβ as based on the on We a results and of Aβ levels in the on Aβ this as the for to secreted Aβ of and in were with and on of Aβ in the was were to and with in for or were for with Aβ or and with or of were with JNK, or was used were by and on a We that the of levels of JNK, ERK, and was by metal in cells. In and other used for protein were to be on metal R. K. M. I. R. A. M. D. R. M. D. G. R. A. A. F. K. J. Q.-X. A. I. and C. L. and protein were by of kinase protein levels protein levels on to was an cell were with CQ, or CQ and Cu2+ or for with a the were by and for an The was and cells were with of addition of for was by on a with and MMP activity of in the and cell was an MMP the or cell were with the The used were is is is and is is and is and was of by the effect of on resulting in increased with and described in from a of were CQ of Cu2+ and Zn2+ in CQ is a metal the effect of CQ on metal levels in cells were with CQ or in the of or for and metal levels were by Cu2+ levels were of and to CQ increased this to of protein Treatment with CQ and Cu2+ a in Cu2+ levels of protein; CQ also increased Zn2+ levels from to of protein of cell revealed significant effect on cell after of to CQ and Cu2+ or Treatment of with resulted in a in with CQ and not Analogous effects of CQ on metal levels were also in cells. increased cell Cu2+ levels by compared with untreated controls. Treatment with Zn2+ levels by in cells with CQ and for metal levels compared with in a CQ and Cu2+ or Zn2+ Aβ in CQ Aβ in cells. Treatment of with CQ for significant to Aβ-(1–40) levels in the Cu2+ for a in Aβ-(1–40) levels were to CQ and a reduction of secreted Aβ-(1–40) levels An effect was treatment with CQ and Zn2+ significant were in Aβ-(1–40) levels cells were with CQ of secreted Aβ-(1–42) levels also with cells as Aβ-(1–42) levels in cells were the of the ELISA, subsequent of Aβ was to Aβ-(1–40). The loss of secreted Aβ treatment with was by of the and mass not of Aβ by of CQ and the of CQ in secreted Aβ with CQ with or Cu2+ for Aβ-(1–40) was significantly and CQ Cu2+ We also the effects of of CQ on Cu2+ in cells. CQ an of in Cu2+ levels CQ resulted in of Cu2+ compared with levels The of of CQ to Cu2+ levels with the reduction of secreted Aβ levels by CQ has been to Cu2+ a of M. C. P. L. P. 2004; PubMed Scopus Google studies significant in Cu2+ and of Aβ levels the the effects of Cu2+ on secreted Aβ were to CQ with of Cu2+. Cu2+ significantly Aβ levels of Cu2+ secreted Aβ levels We also the of Aβ by CQ Cu2+ We an in Aβ levels from to after addition of loss of Aβ was from to after treatment of metal levels revealed a in Cu2+ after a to Cu2+ levels increased a of of Aβ by with Aβ is a loss in to CQ or to and as shown in secreted Aβ was a reduction in the of in were was with that the activity of in was after treatment with of cells with a Masters C.L. J. PubMed Scopus Google revealed effect on of by findings that the loss of secreted Aβ treatment with is to from of Aβ by through of JNK and can pathways Kim Y. J. Neurosci. Res. PubMed Scopus Google Scholar, R. D. 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JNK phosphorylation is by of reactive in the cells were to together with the reactive or the Treatment with these not JNK phosphorylation or Aβ loss in is with Zn2+ effects to of as Zn2+ is a metal and not reactive the results that activation of JNK by is not through oxidative of Aβ by of the of a of and Aβ production in cells C.A. 2003; PubMed Scopus Google Scholar). the is with the loss of Aβ production in cells. Treatment of cells with CQ and Cu2+ for resulted in significant activation of of with the phosphorylation by and significantly the in secreted Aβ levels Treatment of with for increased the of and this effect was by was also a small in levels in which may for the increased levels of We activation is of Treatment of with not phosphorylation of as as phosphorylation of JNK and treatment of with of JNK and phosphorylation and not phosphorylation not that activation is of JNK and of the and JNK for of of and JNK pathways the loss of Aβ by up-regulation of these pathways also results in loss of Aβ in cells. to Cu2+ for revealed activation of and Cu2+ also phosphorylation of and JNK with the for revealed activation of JNK not of these treatments or secreted Aβ that the and JNK pathways for the loss of Aβ in cells. of JNK in with suggested that phosphorylation of in cells may JNK with in the of of In the of increased levels compared with effect on that the effect was not through increased and potentiated JNK phosphorylation in with was to the action of or on JNK phosphorylation of JNK phosphorylation by also the of to Aβ loss in the the effect of phosphorylation on JNK with CQ and Cu2+ in the of or increased phosphorylation of and JNK compared with The increased and JNK phosphorylation with a of Aβ levels in the results evidence that increased phosphorylation of in activation of JNK and to loss of secreted of of cells to for resulted in with cell of an role for or oxidative stress of cells to a matrix after treatment with CQ and Cu2+. shown in cell to by The loss of be by treatment with or loss of cell is with activation of metalloproteases R. PubMed Scopus Google cells with metalloprotease and MMP significantly loss of cell to metalloproteases Aβ cells were with a of metalloprotease and Aβ levels were after to metalloprotease significantly the in secreted Aβ levels by that the loss of secreted Aβ was through increased cell were to synthetic Aβ-(1–40) for with or of Aβ-(1–40) levels in the revealed in the after clearance by cell and/or of to CQ or Cu2+ increased the levels of synthetic Aβ-(1–40) in the after treatment of with significantly Aβ-(1–40) levels by compared with controls and by compared with CQ this effect was by of with or of metalloproteases The results a role for metalloprotease of Aβ as the of Aβ loss in with of and MMP-3 through of the and JNK of and MMP loss of Aβ and cell a role for up-regulation of in the activity of in cells with MMP of cell or the after treatment with for revealed a significant of the of and MMP-3 significant were in the of and of cell with to and the results from the and of were in to revealed a activation of and MMP-3 by were with MMP of with activation of by significant effect on MMP-3 activity MMP-3 activation of not activation by An effect on or MMP-3 activation by that and activation of and MMP-3 the of and MMP-3 significantly the loss of Aβ-(1–40) by effects were with a report that and MMP-3 can Aβ several (10Cherny 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 (1321) Google Scholar). In of from revealed Aβ with Aβ of were in suggested that of the Aβ may be in these through activation of was by of Aβ loss by with alternative cell for to Aβ to Treatment of neuroblastoma cells with CQ and Cu2+ for Aβ levels from to CQ or Cu2+ significant effect In and cells were with CQ and Cu2+ together with synthetic Aβ-(1–40). of Aβ levels in the after revealed significantly synthetic Aβ-(1–40) levels in all cell after treatment with and this be by with results that can secreted Aβ levels via metalloproteases in cell neuroblastoma cells. In this have shown for the that the metal ligand CQ secreted Aβ levels in treatment of cells with CQ effect on Aβ levels in the treatment with CQ to Cu2+ or Zn2+ extracellular Aβ We have shown that this effect is to the of CQ to in Cu2+ or Zn2+ resulting in up-regulation of MMP that of CQ or Cu2+ induce a significant loss of Aβ after The with which Aβ the potential of recent plasma levels of CQ small phase clinical trials (8Ritchie 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 (900) Google Scholar). CQ levels in the may of which to Y. K. M. Arch. PubMed Scopus Google Scholar). were the of CQ levels to Aβ in Cu2+ or Zn2+ was Cu2+ levels can from in the extracellular of the to in the Zn2+ is also in the with levels Bush A.I. Nat. Rev. Neurosci. 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Res. 2004; PubMed Scopus Google which is with findings in cells. and JNK phosphorylation was to induce the in secreted Aβ that Cu2+ has a effect on cell resulting in up-regulation of a of of and and that be to CQ and Aβ can be degraded in vitro and in vivo by several insulin-degrading enzyme, and (4Roher A.E. Kasunic T.C. Woods A.S. Ball M.J. Fridman R. Biochem. Biophys. Res. Commun. 1994; 205: 1755-1761Crossref PubMed Scopus (124) Google Scholar, 6Backstrom J.R. Lim G.P. Cullen M.J. Tokes Z.A. J. Neurosci. 1996; 16: 7910-7919Crossref PubMed Google Scholar, X. X. 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J. 2001; Full Text Full Text PDF PubMed Scopus Google and proteases also in the loss of Aβ by is not be to the Aβ in and to identify and is a in the clearance of secreted CQ of Aβ in vivo is not MMP and in AD and up-regulation of MMP activity in to Aβ in vitro S. J. Neurosci. Res. PubMed Scopus Google Scholar). this may from also be an to Aβ recent studies have shown that in central Cu2+ levels in Aβ levels and deposition S. A. A. R. A. K. C. D. M. Multhaup G. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar, S. V. Y. P. Yang J. J. J. R.A. P. D. Proc. Natl. Acad. Sci. U. S. A. 2003; PubMed Scopus Google Scholar). Cherny (10Cherny 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 (1321) Google demonstrated that mice with CQ have central Cu2+ and Zn2+ levels together with Aβ reports with findings that Cu2+ or Zn2+ levels can Aβ levels by Aβ small phase clinical trials of CQ demonstrated plasma Aβ-(1–42) levels with plasma Zn2+ levels in patients (8Ritchie 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 (900) Google Scholar). increased of Aβ through Zn2+ this the of Aβ with metal as a of the Aβ In studies a potentially important therapeutic role for of MMP activation by metal and subsequent Aβ CQ also clearance of Aβ in vivo through activation of Aβ-degrading MMPs, these findings have important for the of AD based on modulation of metal bioavailability. We for the and for the cells.