Copper levels are increased in the cerebral cortex and liver of APP and APLP2 knockout mice

Brain Research 842 Ž1999. 439–444 www.elsevier.comrlocaterbres

Research report

Copper levels are increased in the cerebral cortex and liver of APP and APLP2 knockout mice Anthony R. White a,b , Rosario Reyes c,d , Julian F.B. Mercer c,d , James Camakaris e , Hui Zheng f , Ashley I. Bush a,b,g , Gerd Multhaup h , Konrad Beyreuther h , Colin L. Masters a,b , Roberto Cappai a,b,) a

d

Department of Pathology, The UniÕersity of Melbourne, ParkÕille, Victoria, 3052 Australia b The Mental Health Research Institute, ParkÕille, Victoria, 3052 Australia c The Scobie and Claire Mackinnon Trace Elements Laboratory, Murdoch Institute, Royal Children’s Hospital, ParkÕille, Victoria, 3052 Australia Centre for Cellular and Molecular Biology, School of Biological and Chemical Sciences, Burwood Campus, Deakin UniÕersity, Burwood, Victoria, 3125 Australia e Department of Genetics, The UniÕersity of Melbourne, ParkÕille, Victoria, 3052 Australia f Department of Genetics and Molecular Biology, Merck Research Laboratories, Rahway, NJ 07065, USA g Department of Psychiatry, and Genetics and Aging Unit, HarÕard Medical School, Massachusetts General Hospital, Charlestown, MA 02129, USA h Center for Molecular Biology, The UniÕersity of Heidelberg, Im Neuenheimer Feld 282, 69120 Heidelberg, Germany Accepted 13 July 1999

Abstract The pathological process in Alzheimer’s disease ŽAD. involves amyloid beta ŽAb . deposition and neuronal cell degeneration. The neurotoxic Ab peptide is derived from the amyloid precursor protein ŽAPP., a member of a larger gene family including the amyloid precursor-like proteins, APLP1 and APLP2. The APP and APLP2 molecules contain metal binding sites for copper and zinc. The zinc binding domain ŽZnBD. is believed to have a structural rather than a catalytic role. The activity of the copper binding domain ŽCuBD. is unknown, however, APP reduces copper ŽII. to copper ŽI. and this activity could promote copper-mediated neurotoxicity. The expression of APP and APLP2 in the brain suggests they could have an important direct or indirect role in neuronal metal homeostasis. To examine this, we measured copper, zinc and iron levels in the cerebral cortex, cerebellum and selected non-neuronal tissues from APP ŽAPPyry. and APLP2 ŽAPLP2yry. knockout mice using atomic absorption spectrophotometry. Compared with matched wild-type ŽWT. mice, copper levels were significantly elevated in both APPyry and APLP2yry cerebral cortex Ž40% and 16%, respectively. and liver Ž80% and 36%, respectively.. Copper levels were not significantly different between knockout and WT cerebellum, spleen or serum samples. There were no significant differences observed between APPyry, APLP2yry and WT mice zinc or iron levels in any tissue examined. These findings indicate APP and APLP2 expression specifically modulates copper homeostasis in the liver and cerebral cortex, the latter being a region of the brain particularly involved in AD. Perturbations to APP metabolism and in particular, its secretion or release from neurons may alter copper homeostasis resulting in increased Ab accumulation and free radical generation. These data support a novel mechanism in the APPrAb pathway which leads to AD. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Metals; Amyloid precursor protein; Copper; Oxidative stress; Knockout

1. Introduction Alzheimer’s disease ŽAD. is characterized by progressive neurodegeneration associated with extracellular amyloid formation and the appearance of neurofibrillary tangles w37x. The amyloid plaques consist primarily of a

) Corresponding author. Department of Pathology, University of Melbourne, Parkville, Victoria, 3052 Australia. Fax: q61-3-9344-4004; E-mail: [email protected]

39–43 amino acid peptide, amyloid beta ŽAb ., that undergoes a conformational change and aggregation to form fibrillar and amorphous deposits w12,22x. The role of Ab in AD is postulated to involve neuronal degeneration as supported by the in vitro neurotoxicity of Ab w19,41x. Ab is derived by proteolytic cleavage of the Alzheimer amyloid precursor protein ŽAPP., a member of a multigene family including the amyloid-like precursor proteins ŽAPLP1 and APLP2. w17,36,39x. APP and APLPs share a number of motifs and functions such as heparin and metal binding domains and neurotrophic activity w4,7,8,16,24–

0006-8993r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 0 6 - 8 9 9 3 Ž 9 9 . 0 1 8 6 1 - 2

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A.R. White et al.r Brain Research 842 (1999) 439–444

26,34x. APP may also have a neuroprotective effect as the addition of secreted APP ŽsAPP. to culture medium or the overexpression of transfected human APP protects cells from neurotoxic insults w23,28,29,35x. Recent evidence also suggests that APP may have a role in cell adhesionrsignalling pathways and arachidonic acid metabolism w10,15x. Perturbed APP metabolism or expression levels, however, may result in neurotoxic processes w24,26,27x. One domain that could contribute to such activities is the copper binding domain ŽCuBD. residing between residues 135 and 158 of APP and APLP2 Žbut not APLP1. w16,26,27x. This sequence is capable of binding and reducing copper ŽII. to copper ŽI.. We have recently found that APP and peptides encoding the CuBD can modulate copper-mediated lipid peroxidation and neurotoxicity in cultures Žmanuscript submitted.. APP, APLP2 and APLP1 also have a zinc binding domain ŽZnBD. which is believed to have a structural role rather than a direct catalytic function. Due to the highly toxic potential of copper-generated free radicals such as the hydroxyl radical ŽOH Ø ., copper levels are strictly regulated in all tissues through the expression of copper binding proteins w20x. The relative abundance of the APP and APLP2 proteins in brain tissue therefore suggests an important direct or indirect role for these proteins in metal metabolism andror homeostasis. It has recently been shown that ablation of the prion protein ŽPrP. Ža neuronal cuproprotein. decreases brain copper levels w6x. To determine whether APP expression could affect metal biostasis in vivo, we examined copper, zinc and iron levels in brain and selected non-neuronal tissues from APP knockout ŽAPPyry . and wild-type ŽWT. control mice using atomic absorption spectrophotometry ŽAAS.. Our study shows that adult APPyry mice have significantly increased copper but not zinc or iron levels in the cerebral cortex and liver compared to age- and genetically-matched WT mice. APLP2yry mice also revealed increases in copper in cerebral cortex and liver. These findings indicate the APP family can modulate copper homeostasis and suggests that APPrAPLP2 expression may be involved in copper efflux from liver and cerebral cortex.

Instruments for tissue isolation were washed in 0.02 M EDTA prior to use. Upon removal, samples were washed in saline solution and frozen at y708C prior to analysis. 2.3. Metal analysis 2.3.1. Analysis of copper, zinc and iron in mouse tissues Dried tissue samples were transferred to acid-washed 10 ml plastic tubes, dissolved in 0.5 ml concentrated nitric acid at room temperature ŽRT. for 1 h and placed in a pre-heated block at 658C for 4 h. Subsequently, the tube was vortexed, diluted with 2.5 ml of deionized H 2 O, vortexed again and centrifuged for 5 min at 1500 rpm. The supernatant was transferred to a fresh 10 ml tube and analysed by direct aspiration into the flame of an atomic absorption spectrophotometer ŽVARIAN SpectrAA-880.. Metal levels are expressed as mgrg tissue Ždry weight.. 2.3.2. Analysis of copper in mouse sera A direct measurement of copper levels in sera was made using the Graphite Tube Atomiser ŽGTA 100.. The sera were pre-diluted to suit the calibration range of the GTA. 20 ml of sample was pipetted into a 1.5 ml acidwashed Eppendorf tube, diluted with 980 ml of 0.5% nitric acid, capped and vortex mixed. The sample was transferred to an ultra-pure washed GTA cup and analysed using a Programmable Sample Dispenser. Copper levels are expressed as mmolrl sera. 2.4. Statistical analysis Metal levels were measured in 6 to 20 mice of each type ŽAPPyry, APLP2yry or WT mice.. Cerebral cortex, cerebellum and liver samples were determined in two to three separate groups of mice with tissues isolated and measured at different times from each group. Spleen and sera samples were taken from one or two separate groups of mice. Data are presented as mean and standard deviations from the mean. Data was analysed using ANOVA and Newman–Keuls tests for significance.

3. Results 2. Materials and methods 2.1. Mice Generation and initial characterization of APPyry and APLP2yry mice has been previously described w38,42x. Control WT mice were the same strain as both APPyry and APLP2yry mice ŽC57BL6Jx129sv.. Tissues and sera were taken from age- and gender-matched mice. 2.2. Tissue preparation To minimize metal contamination of samples, all tubes were acid washed with 20% nitric acid prior to use.

3.1. Cerebral cortex from APP yry and APLP2 yry mice reÕeals increased copper leÕels compared to WT mice The presence of zinc and copper binding sequences in the APP ectodomain w7,16,26x suggests a possible role for this protein in metal homeostasis. To investigate this, we measured zinc, copper and iron levels in cerebral cortex and cerebella taken from adult Ž9–10 week old. APPyry and WT mice using AAS. Copper levels were found to be 12.7 " 1.3 mgrg tissue Ždry weight. in cerebral cortex from WT mice. In contrast, the copper level in cerebral cortex from age and genetically matched APPyry mice

A.R. White et al.r Brain Research 842 (1999) 439–444 Table 1 Metal concentration in mouse cerebral cortex Copper, zinc and iron levels were determined in cerebral cortices taken from adult APPyry, APLP2yry and matched WT mice using atomic absorption spectrophotometry. Metal levels are given in mgrg tissue Ždry weight.. Mean copper levels were significantly higher in APPyry Ž a p0.01. and APLP2yry Ž b p- 0.05. cerebral cortices as compared to WT mice. Copper levels were also significantly higher in APPyry cortices than in APLP2yry cortices Ž c p- 0.01.. No significant difference in zinc or iron levels was observed between APPyry, APLP2yry or WT cerebral cortices. Mouse

APPqrq APPyry APLP2yry

n value

ns 20 ns13 ns8

Metal concentration Žmgrg tissue dry weight.

Table 2 Metal concentration in mouse cerebellum Copper, zinc and iron levels were determined in cerebella taken from adult APPyry, APLP2yry and matched WT mice using atomic absorption spectrophotometry. Metal levels are given in mgrg tissue Ždry weight.. Mean copper levels were not significantly different between APPyry, APLP2yry or WT cerebella. No significant difference in zinc or iron levels was observed between APPyry, APLP2yry or WT cerebella. Mouse

qrq

Copper

Zinc

Iron

12.7"1.3 17.7"2.3 a,c 14.8"1.5 b

70.7"3.6 72.9"7.2 72.5"7.6

69.1"4.6 71.4"8.0 60.0"12.8

was approximately 40% higher at 17.7 " 2.3 mgrg Ž p 0.01, Table 1.. The zinc and iron levels in the same tissue samples revealed no significant differences in the level of either metal between APPyry and WT mice ŽTable 1.. The distribution of copper levels in individual mouse cerebral cortices shows that WT mice had copper levels between 11 and 15 mgrg tissue ŽFig. 1.. In contrast, the majority Ž10r13. of APPyry cerebral cortex copper levels were between 16 and 20 mgrg tissue, however, some mice Ž3r13. had lower levels of 12 and 13 mgrg tissue ŽFig. 1. indicating that other factors may influence copper levels in

441

APP APPyry APLP2yry

n value

ns11 ns8 ns8

Metal concentration Žmgrg tissue dry weight. Copper

Zinc

Iron

16.4"2.9 17.8"3.1 17.1"3.7

61.2"6.0 60.0"9.7 61.7"7.4

73.3"14.3 64.1"12.3 64.3"11.4

APPyry cerebral cortex. Metal levels were also examined in cerebella taken from the same animals and no significant differences in the concentrations of any metal were observed between APPyry and WT mice ŽTable 2.. These data demonstrate that APP expression can significantly and specifically affect copper levels in the cerebral cortex, but not cerebella, of adult mice. The APLP2 molecule also has zinc and copper binding domains and could potentially contribute to metal homeostasis. Examination of copper levels in cerebral cortex from APLP2yry and WT mice revealed a 16% increase in copper in APLP2yry samples Ž p - 0.05, Table 1.. This

Fig. 1. The range of copper levels in the cerebral cortex of APPyry, APLP2yry and WT mice. Copper levels were determined in the cerebral cortex of adult APPyry, APLP2yry and genetic and age-matched WT mice using atomic absorption spectrophotometry. Copper levels are given as mgrg tissue Ždry weight.. WT mice revealed copper levels in the range of 11–15 mgrg tissue. Most APPyry cortices had copper levels in the range of 18 to 20 mgrg tissue, however, several mice revealed low copper levels of between 12 and 16 mgrg. The majority of APLP2yry cerebral cortex copper levels were between 14 and 17 mgrg tissue. These data show that APP expression can modulate copper levels in the cerebral cortex of adult mice, although other factors are also involved.

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442

Table 3 Metal concentration in mouse liver Copper, zinc and iron levels were determined in liver taken from adult APPyry, APLP2yry and matched WT mice using atomic absorption spectrophotometry. Metal levels are given in mgrg tissue Ždry weight.. Mean copper levels were significantly higher in APPyry and APLP2yry liver as compared to WT mice Ž a p- 0.01.. No significant difference in zinc or iron levels was observed between APPyry, APLP2yry or WT liver. Mouse

qrq

APP APPyry APLP2yry

n value

ns6 ns6 ns6

Metal concentration Žmgrg tissue dry weight. Copper

Zinc

Iron

13.4"4.6 24.3"8.9 a 18.2"2.5a

100"30.3 112"19.2 103"9.0

224"60 212"35 209"42

level Ž14.8 " 1.5 mgrg tissue. was significantly lower than the APPyry cerebral cortex Ž p - 0.01.. The majority Ž7r8. of APLP2yry cerebral cortex copper levels were between 14 and 17 mgrg tissue ŽFig. 1.. Consistent with APPyry mice, no difference was observed in the zinc or iron levels between APLP2yry and WT mouse cerebral cortex or in any metal level in cerebella from these mice ŽTables 1 and 2.. 3.2. Copper leÕels are increased in the liÕer of APP yry and APLP2 yry compared to WT mice To determine if APP expression also affects copper levels in non-neuronal tissues, metal concentrations were measured in liver and spleen taken from adult APPyry and WT mice. The APPyry mice had 24.3 " 8.9 mg copperrg tissue compared to 13.4 " 4.6 mgrg tissue in WT mice Ž p - 0.01, Table 3., an increase of approximately 80%. There was no significant difference in liver zinc or iron levels between APPyry and WT mice ŽTable 3. indicating that the increased copper level is not due to increased blood contamination in the APPyry samples. There were no significant differences in spleen metal levels ŽTable 4. indicating a specificity for increased copper levels in

Table 4 Metal concentration in mouse spleen Copper, zinc and iron levels were determined in spleen taken from adult APPyry, APLP2yry and matched WT mice using atomic absorption spectrophotometry. Metal levels are given in mgrg tissue Ždry weight.. Mean copper levels were not significantly different between APPyry, APLP2yry or WT spleen. No significant difference in zinc or iron levels was observed between APPyry, APLP2yry or WT spleen. Mouse

n value

APPqrq APPyry APLP2yry

ns8 ns8 ns6

Metal concentration Žmgrg tissue dry weight. Copper

Zinc

Iron

5.0"1.6 4.7"1.5 5.2"0.7

82.1"13.5 80.0"4.4 85.2"15.3

2176"991 2010"521 1595"672

Table 5 Copper concentration in mouse sera Copper levels were determined in sera taken from adult APPyry, APLP2yry and matched WT mice using atomic absorption spectrophotometry. Metal levels are given in mmolrl sera. Mean copper levels were not significantly different between APPyry, APLP2yry or WT sera. Mouse qrq

APP APPyry APLP2yry

n value

Copper concentration Žmmolrl sera.

ns6 ns6 ns6

12.1"6.5 9.2"4.0 10.5"0.8

APPyry liver. Examination of copper levels in sera revealed a small but not statistically significant difference between APPyry and WT mice Ž p - 0.362, Table 5. suggesting that the increased copper levels in brain and liver were not caused by deposition from excessive levels in the blood. Analysis of metal in liver samples from APLP2yry mice revealed significantly higher levels of copper compared to WT mice Ž18.2 " 2.5 and 13.4 " 4.6 mgrg tissue for APLP2yry and WT liver, respectively. Ž p - 0.01, Table 3.. No differences were observed in zinc or iron levels between APLP2yry and WT liver ŽTable 3. and no difference in any metal level was found in the spleen or sera of APLP2yry and WT mice ŽTables 4 and 5.. These findings provide further evidence that the copper binding domain expressed in the APP family is important for copper homeostasis in both liver and cerebral cortex in adult mouse.

4. Discussion The normal biological function of the amyloid precursor family is not well understood. The identification of zinc and copper metal binding sites on these proteins suggests a possible role in biometal homeostasis w7,16,25–27x. To test this hypothesis, the levels of copper and other metals in the brain and selected non-neuronal tissues from mice devoid of APP or APLP2 were measured. This analysis revealed that APP and APLP2 expression significantly and specifically lowers copper levels in adult mouse cerebral cortex and liver. The increased copper levels in cerebral cortex and liver of the knockout mice are not an artefact of the knockout procedure as copper levels were significantly different between APLP2yry and APPyry cerebral cortex. Furthermore, copper levels were lower in the cerebral cortex of PrP knockout ŽPrPyry . mice w6x. The PrPyry mice were of the same background as the APP, APLP2 and WT mice, indicating the effect is protein specific. In addition, the WT mouse copper and zinc levels were in close agreement with previously reported levels for adult mouse cerebral cortex, cerebella and liver, regardless of mouse strain w18,31x. The changes in copper levels in

A.R. White et al.r Brain Research 842 (1999) 439–444

APPyry mice were not associated with the loss of the metal binding activity of Ab, as rodent Ab does not bind significant levels of copper or zinc w1x. APLP2 also contains a copper binding ectodomain, however, APLP2 had a smaller effect than APP on copper levels in cerebral cortex. The differences between copper levels in the liver of APPyry and APLP2yry mice were not significant. The reason for the difference in the cerebral cortex copper levels could relate to lower expression or a different distribution of APLP2 to APP or the influence of other residues outside of the copper binding sequence w26x. This is consistent with APLP2 having only 60% of the copper redox activity of APP w26x. The redox activity may be critical to the ability of APP and APLP2 to transport copper and is supported by the finding that copper ŽII. reduction modulates copper uptake in eukaryotic cells w14x. Accumulating evidence suggests that biometals such as iron, zinc and copper may play an important role in several neurodegenerative disorders w5,9,21,30x. In particular, copper has been shown to bind or interact with proteins central to neurodegeneration in AD, transmissible spongiform encephalopathies Žprion protein., amyotrophic lateral sclerosis Žcopperrzinc superoxide dismutase. and Parkinson’s disease Žmonoamine oxidase. w1,6,11,16,26,30x. As copper has a high capacity to generate free radicals, even at low concentrations, copper metabolism must be tightly regulated to prevent tissue damage w13,20x. Neurons have relatively low concentrations of anti-oxidants compared to other cell-types w2x and strict copper regulation is therefore crucial to prevent neuronal degeneration. As a consequence, abundant cell surface proteins such as APP and APLP2 that contain a copper binding sequence may have an important role in copper metabolism andror homeostasis. Although this study clearly demonstrates that APP and APLP2 are involved in modulating copper but not iron or zinc levels in specific tissues in adult mice, it is not clear how this process occurs or which tissue or fluid is the predominant site of interaction between copper and APP or APLP2. Cells in brain and liver express relatively high levels of APP w3x and the release of cellular APPrAPLP2 with bound copper may provide a means of removing excess copper from certain cell-types. This study suggests that perturbations to APP metabolism could initiate pathological changes associated with copper metabolism. We have found that WT neurons are more vulnerable than APPyry neurons to physiological concentrations of copper but not other metals. The WT neurons had increased levels of lipid peroxidation products and similar effects were obtained with a peptide containing the APP CuBD Žmanuscript submitted.. These data support a copper-APP association that is relevant to AD. Furthermore, primary neurons exposed to Ab reveal decreased sAPP levels and significantly increased cell-associated APP w32,33,40x. Decreased sAPP could raise copper levels in the cerebral cortex which in turn, promotes human Aß

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aggregation w1x as well as increased free radical production from Aß andror cell-associated APP w5,27x. Due to the highly conserved sequence homology of the CuBD between species it is very likely that APP has a similarly important role in copper homeostasis in humans. Perturbation of this function could have important implications for disease initiationrprogression since the cortex is commonly affected in AD. Finally, this study adds to the growing body of data w1,5,21,26,27x suggesting an important role for copper in AD pathogenesis. Acknowledgements We would like to thank Dr. Sangram Sisodia and Dr. Connie von Koch for the APLP2yry mice and Mr. Ilia Voskoboinik for helpful discussions. This work is supported in part by grants from the National Health and Medical Research Council of Australia to C.L.M. K.B. is supported by the Deutshe Forschungsgemeinschaft and the Bundesministerium fur Forschung und Technologie. References w1x C.S. Atwood, R.D. Moir, X.D. Huang, R.C. Scarpa, N.M.E. Bacarra, D.M. Romano, M.K. Hartshorn, R.E. Tanzi, A.I. Bush, Dramatic aggregation of Alzheimer A-beta by Cu ŽII. is induced by conditions representing physiological acidosis, J. Biol. Chem. 273 Ž1998. 12817–12826. w2x J.S. Bains, C.A. Shaw, Neurodegenerative disorders in humans: the role of glutathione in oxidative stress-mediated neuronal death, Brain Res. Rev. 25 Ž1997. 335–358. w3x J. Beer, C.L. Masters, K. Beyreuther, Cells from peripheral tissues that exhibit high APP expression are characterized by their high membrane fusion activity, Neurodegeneration 4 Ž1995. 51–59. w4x D. Beher, L. Hesse, C.L. Masters, G. Multhaup, Regulation of amyloid protein precursor ŽAPP. binding to collagen and mapping of the binding sites on APP and collagen type I, J. Biol. Chem. 271 Ž1996. 1613–1620. w5x S.C. Bondy, S.X. Guo-Ross, A.T. Truong, Promotion of transition metal-induced reactive oxygen species formation by b-amyloid, Brain Res. 799 Ž1998. 91–96. w6x D.R. Brown, K. Qin, J.W. Herms, A. Madlung, J. Manson, R. Strome, P.E. Fraser, T. Kruck, A. Bohlens, W. Schultz-Schaeffer, A. Giese, D. Westaway, H.A. Kretzschmar, The cellular prion protein binds copper in vivo, Nature 390 Ž1997. 684–687. w7x A.I. Bush, G. Multhaup, R.D. Moir, T.G. Williamson, D.H. Small, B. Rumble, P. Pollwein, K. Beyreuther, C.L. Masters, A novel zincŽII. binding site modulates the function of the beta A4 amyloid protein precursor of Alzheimer’s disease, J. Biol. Chem. 268 Ž1993. 16109–16112. w8x R. Cappai, S.S. Mok, D. Galatis, D.F. Tucker, A. Henry, K. Beyreuther, D.H. Small, C.L. Masters, Recombinant human amyloid precursor-like protein 2 ŽAPLP2. expressed in the yeast Pichia pastoris can stimulate neurite outgrowth, FEBS Lett. 442 Ž1999. 95–98. w9x M.A. Deibel, W.D. Ehmann, W.R. Markesbery, Copper, iron, and zinc imbalances in severely degenerated brain regions in Alzheimer’s disease: possible relation to oxidative stress, J. Neurol. Sci. 143 Ž1996. 137–142. w10x A. Fossgreen, B. Bruckner, C. Czech, C.L. Masters, K. Beyreuther, R. Paro, Transgenic Drosophila expressing human amyloid precursor

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