Gene knockout of amyloid precursor protein and ... - Wiley Online Library

Journal of Neurochemistry, 2004, 91, 423–428

doi:10.1111/j.1471-4159.2004.02731.x

Gene knockout of amyloid precursor protein and amyloid precursor-like protein-2 increases cellular copper levels in primary mouse cortical neurons and embryonic fibroblasts Shayne A. Bellingham,* Giuseppe D. Ciccotosto, B. Elise Needham, Lisa R. Fodero, Anthony R. White, Colin L. Masters, Roberto Cappai ,à and James Camakaris* *Department of Genetics, Department of Pathology and àCentre for Neuroscience, The University of Melbourne, Victoria, Australia

Abstract Alzheimer’s disease is characterised by the accumulation of amyloid-b peptide, which is cleaved from the copper-binding amyloid-b precursor protein. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer’s disease neuropathogenesis and suggested a role for amyloid-b precursor protein and amyloid-b in copper homeostasis. Amyloid-b precursor protein is a member of a multigene family, including amyloid precursor-like proteins-1 and -2. The copper-binding domain is similar among amyloid-b precursor protein family members, suggesting an overall conservation in its function or activity. Here, we demonstrate that double knockout of amyloid-b precursor protein and amyloid precursor-like protein-2 expression

results in significant increases in copper accumulation in mouse primary cortical neurons and embryonic fibroblasts. In contrast, over-expression of amyloid-b precursor protein in transgenic mice results in significantly reduced copper levels in primary cortical neurons. These findings provide cellular neuronal evidence for the role of amyloid-b precursor protein in copper homeostasis and support the existing hypothesis that amyloid-b precursor protein and amyloid precursor-like protein-2 are copper-binding proteins with functionally interchangeable roles in copper homeostasis. Keywords: Alzheimer’s disease, amyloid precursor-like protein 2, amyloid precursor protein, cortical neurons, gene knockout-out mice, neuronal copper homeostasis. J. Neurochem. (2004) 91, 423–428.

The amyloid-b precursor protein (APP) of Alzheimer’s disease is a type 1 transmembrane cuproprotein. The proteolytic processing of APP by secretases yields the amyloid-b peptide (Ab), the primary constituent of the amyloid plaque (Haass and Selkoe 1993). APP is a member of a multigene family that contains the paralogues amyloid precursor-like protein 1 and 2 (APLP1 and APLP2) (Wasco et al. 1992, 1993). Orthologues have been identified in a diverse range of species including Drosophila melanogaster, Xenopus laevis and Caenorhabditis elegans (Rosen et al. 1989; Okado and Okamoto 1992; Daigle and Li 1993). APP has a primary copper-binding domain, located in the N-terminal cysteinerich region next to the growth factor-like domain, and a secondary copper-binding domain, which is generated in Ab after proteolytic processing of APP (Hesse et al. 1994; Atwood et al. 2000). Both APP and Ab can strongly bind copper, as Cu(II), and reduce it to Cu(I) in vitro (Multhaup et al. 1996; Huang et al. 1999). Elevated copper concentrations reduce Ab production and increase secretion of APP in a cell line transfected with human APP cDNA (Borchardt et al.

1999). More recently, reports show that increased brain copper levels cause a decrease in Ab production in APP transgenic mouse models in vivo (Bayer et al. 2003; Phinney et al. 2003), while severely depleted cellular copper decreases APP gene expression (Bellingham et al. 2004). The copper-binding region is well conserved amongst the different APP-gene family members (Simons et al. 2002;

Received June 11, 2004; revised manuscript received June 29, 2004; accepted June 29, 2004. Address correspondence and reprint requests to Assoc. Prof. James Camakaris, Department of Genetics, The University of Melbourne, Parkville, Victoria, AUSTRALIA 3010. E-mail: j.camakaris@unimelb. edu.au or Dr Roberto Cappai, Department of Pathology, The University of Melbourne, Parkville, Victoria, AUSTRALIA 3010. E-mail: [email protected] Abbreviations used: Ab, amyloid-b peptide; APLP1, amyloid precursor-like protein-1; APLP2, amyloid precursor-like protein-2; APP, amyloid-b precursor protein; DMEM, Dulbecco’s minimal Eagle’s medium; HBSS, Hanks’ balanced salt solution; MEF, mouse embryonic fibroblast; MEM, minimal essential medium; NTg, non-transgenic.

Ó 2004 International Society for Neurochemistry, J. Neurochem. (2004) 91, 423–428

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White et al. 2002). In vitro studies, using primary neuronal cultures derived from APP knockout mice, showed that the APP copper-binding domain can either promote or inhibit Cu(I)-mediated neurotoxicity in an orthologue sequencedependent manner (White et al. 2002). In vivo mouse studies have provided compelling evidence that the APP and APLP2 proteins are modulators of copper homeostasis. APP and APLP2 knockout mice (APP–/– and APLP2–/–, respectively) have significantly increased copper levels in the brain and liver (White et al. 1999b). Conversely, APP over-expressing transgenic mice have significantly reduced copper levels in transgenic mouse brain (Maynard et al. 2002). The three-dimensional structure of the human APP copper-binding domain (APP residues 124–189) has been determined by NMR spectroscopy (Barnham et al. 2003). Importantly, the copper-binding domain has structural homology to copper chaperones, thus strongly supporting the in vivo data and suggests that the APP copper-binding domain functions as a neuronal metal-transporter and/or metal-chaperone to modulate copper homeostasis. However, the mechanism by which the APP and APLP2 function together in cellular copper homeostasis is unknown. To understand the relationship between APP and APLP2 expression and cellular copper homeostasis, we have utilised primary neuronal cultures from APP–/– and APLP2–/– mice to characterise copper transport in these cells. In addition, we studied copper homeostasis in neuronal cultures derived from APP transgenic mice. Our results provide strong evidence for a functional role of APP in neuronal copper homeostasis by demonstrating for the first time a gene dose-dependant relationship between decreasing APP expression and increasing copper accumulation, whilst over-expression of APP decreased copper accumulation. We also provide evidence for the functional redundancy of APP and APLP2 in modulating neuronal copper homeostasis. These data support the hypothesis that APP and APLP2 are copper proteins that maintain neuronal copper homeostasis.

Experimental procedures Mice The generation and initial characterization of APP–/– and APLP2–/– mice has been previously described (Zheng et al. 1995; von Koch et al. 1997). Control wild-type mice (APP+/+/APLP2+/+) were derived from the same background strain as both APP–/– and APLP2–/– mice (C57BL6J/129sv). Double knockout mice, APP–/–/APLP2–/–, were generated by crossing hemizygous APP+/–/ APLP2–/– mice, resulting in offspring with the genetic makeup of APP–/–/APLP2–/–, APP+/–/APLP2–/– and APP+/+/APLP2–/–. Genetic background of each individual fetal pup was determined by PCR of tail DNA using primer sets as previously described (von Koch et al. 1997). We maintained the APP transgenic (Tg2576) colony (Hsiao et al. 1996) by crossing Tg2576 males with C57BL6/ SJL F1 females, and determined the transgene status by PCR of tail

DNA, using primer sets as described previously (Hsiao et al. 1995). Non-transgenic (NTg) littermates were used as controls. Preparation of primary neuronal cultures Cortical neuronal cultures were prepared as described previously (White et al. 1998). Litters from embryonic day-15 mice were placed in separate dishes before the preparation of cortical neuronal cultures to prevent cross contamination of genetic background of mice. Briefly, cortices were removed, dissected free of meninges and dissociated in 0.025% (w/v) trypsin. Dissociated cells were plated in 12 wells of poly-L-lysine coated sterile 12-well culture plates (Nunc, Napierville, IL, USA) in minimal essential medium (MEM; Life Technologies Inc., Grand Island, NY, USA) supplemented with 10% fetal calf serum (Commonwealth Serum Laboratories, Parkville, Australia) and 5% horse serum (Commonwealth Serum Laboratories). This method of seeding cells resulted in plating of approximately 800 000 cells per well. Cultures were maintained at 37°C in 5% CO2 for 2 h before the plating medium was replaced with NeurobasalÔ growth medium containing B27 supplements (Life Technologies Inc.). This method resulted in cultures highly enriched for neurons as previously described (White et al. 1998, 1999a). After 6 days in culture, the medium was replaced with fresh NeurobasalÔ medium supplemented with B27 lacking antioxidants for copper experiments. Preparation of mouse embryonic fibroblasts (MEFs) MEFs were prepared from embryonic day 14 or 15 mice using sterile conditions. Individual embryos were placed into separate wells of a 6-well dish (Nunc) and the head, liver and gastrointestinal organs were dissected free and the remaining body washed two times in fresh Hanks’ balanced salt solution (HBSS) buffer (Sigma, St Louis, MO, USA) and placed in a new well with fresh 2.5 mL HBSS buffer. The tissue was finely minced with sterile scissors, 0.25% w/v trypsin was added and the cell suspension transferred to a 15-mL tube and placed in a gently shaking 37°C water bath for 20 min. Trypsin inhibitor mix (0.01% w/v LBTI and 0.003% w/v Dnase) was added to dissolve the DNA released from the cells. The cell suspension was then added to a sterile syringe and the contents passed through an 18-gauge syringe, and then triturated 30 times using a Pasteur pipette. The cell suspension was pelleted, and the medium replaced with high glucose Dulbecco’s minimal Eagle’s medium (DMEM; Life Technologies Inc.) supplemented with 2 mM glutamine, 1 mM sodium pyruvate, 100 U/mL penicillin G, 100 lg/mL streptomycin and 10% fetal calf serum, and added to a T175 flask (Nunc) and placed in a 37°C incubator supplemented with 5% CO2. The culture medium was replaced with fresh DMEM after 24 h and then changed every 3–4 days thereafter. MEFs were allowed to incubate in culture for at least 7–10 days before setting up for experiments. Forty thousand cells were added to each well in a 12-well plate (Nunc) and allowed to grow for 2–3 days before commencement of experiments. On the day of copper experiments, medium was replaced with fresh DMEM supplemented with 10% fetal calf serum. Steady-state measurement of 64Cu accumulation Six-day-old primary cortical neuron cultures and 9–13-day-old MEFs were used for 64Cu accumulation assays. For APP–/–/ APLP2–/–, APP+/–/APLP2–/–, APP+/+/APLP2–/– and control wildtype (APP+/+/APLP2+/+) cortical neuron cultures, the medium was


APP and APLP2 function in neuronal copper homeostasis 425

replaced with fresh NeurobasalÔ growth medium with B27 supplements containing 5–10 lCi/mL 64Cu (as CuCl2; ARI, Lucas Heights, NSW, Australia) and ‘no added copper’ (basal, 0.76 lM Cu) or medium with added ‘cold’ CuCl2 to give a total copper concentration of 15.7 and 50 lM and then incubated at 37°C, 5% CO2 for 24 h. For Tg2576 and control NTg primary cortical neurons cultures, the medium was replaced with fresh NeurobasalÔ growth medium with B27 supplements containing 5–10 lCi/mL 64Cu and ‘no added copper’ (basal, 0.76 lM Cu) or medium with added ‘cold’ CuCl2 to give a total copper concentration of 50, 100 and 150 lM and then incubated at 37°C, 5% CO2 for 48 h. For MEFs, the medium was replaced with fresh DMEM with 10% fetal calf serum containing 5–10 lCi/mL 64Cu and ‘no added copper’ (basal) or medium with added ‘cold’ CuCl2 to give a total copper concentration of 15.7, 32 and 50 lM. MEFs were then incubated at 37°C, 5% CO2 for 24 and 48 h. After incubation, primary cortical neurons and MEFs were immediately harvested to obtain total 64Cu accumulated at the end of the time period (four wells per concentration/time point). All cells were washed twice in ice-cold serum-free medium to remove any non-specifically bound 64Cu and harvested by dissolution in 1.5% sodium dodecyl sulfate (SDS), 2 mM EDTA. Cell lysates were scraped and collected. 64Cu was measured in cell lysates using an LKB-Wallac Ultragamma counter. Protein concentration was determined in each cell lysate using a Bio-Rad Protein Assay (Bradford 1976). Copper accumulation was then expressed as pmol Cu per mg protein as previously described (Camakaris et al. 1995). Statistical analysis One-way ANOVA of more than two means followed by Bonferroni’s multiple comparison of mean’s post-test was performed for 64Cu accumulation assays in APP/APLP2 mouse cortical neurons and MEFs. Unpaired t-test of two means was performed for 64Cu accumulation assays in Tg2576 mouse cortical neurons. Statistical analysis was performed using Graphpad Prism3 for Macintosh (GraphPad Software Inc., San Diego, CA, USA). Statistically significant was defined as p < 0.05.

Results

Increased copper accumulation for APP+/–/APLP2–/– and APP–/–/APLP2–/– primary cortical neurons Primary cortical neurons derived from wild-type (APP+/+/ APLP2+/+), APP+/+/APLP2–/–, APP+/–/APLP2–/– and APP–/–/ APLP2–/– mice were investigated for differences in copper accumulation using a radio-copper assay (Fig. 1). No significant difference in copper accumulation was observed between APP+/+/APLP2–/– and wild-type controls (Fig. 1a). However, a significant increase in copper accumulation was observed for both APP+/–/APLP2–/– and APP–/–/APLP2–/– cortical neurons compared with either wild-type control or APP+/+/APLP2–/– neurons (Fig. 1a). A significant difference in copper accumulation also occurred between APP+/–/ APLP2–/– and APP–/–/APLP2–/– cortical neurons (Fig. 1a). In APP+/–/APLP2–/– neurons, copper levels increased by

Fig. 1 Effect of APP and APLP2 gene knockout on copper accumulation in primary mouse cortical neurons. (a) Copper levels were measured, using 64Cu, in mouse primary cortical neurons derived from wild-type (APP+/+/APLP2+/+), APP+/+/APLP2–/–, APP+/–/ APLP2–/– and APP–/–/APLP2–/– knockout mice. Primary neuronal cultures were incubated in medium containing 64Cu and ‘no added copper’ (basal, 0.76 lM Cu) or medium with added ‘cold’ CuCl2 to give a total copper concentration of 15.7, and 50 lM for 24 h. No significant difference in copper accumulation was observed between wild-type (APP+/+/APLP2+/+) and APLP2 knockout (APP+/+/APLP2–/–) neurons. Significantly increased copper accumulation was observed between wild-type (APP+/+/APLP2+/+) and APP+/–/APLP2–/– neurons; between wild-type (APP+/+/APLP2+/+) and APP–/–/APLP2–/– neurons; between APP+/+/APLP2–/– and APP+/–/APLP2–/– neurons; between APP+/–/APLP2–/– and APP–/–/APLP2–/– neurons; and between APP+/–/APLP2–/– and APP–/–/APLP2–/– neurons. Values for copper accumulation are expressed as pmol Cu/mg protein. Bars represent the mean ± SE from four independent triplicate experiments (ANOVA, Bonferroni’s post test; NS, p > 0.05 compared with APP+/+/APLP2+/+ *p < 0.001 compared with APP+/+/APLP2+/+ neurons; ^p < 0.001 compared with APP+/+/APLP2–/– neurons; #p < 0.001 compared with APP+/–/APLP2–/– neurons). (b) Percentage copper accumulation in APP/APLP2 knockout mouse primary cortical neurons. The mean copper accumulation from APLP2 knockout (APP+/+/APLP2–/–) neurons was used to define 100% copper accumulation. Bars represent the mean ± SE from four independent triplicate experiments (ANOVA, Bonferroni’s post test; ***p < 0.001 compared with APP+/+/APLP2–/– neurons).

55 ± 1% in basal (0.76 lM) Cu medium, 60 ± 5% in 15.7 lM Cu medium, and 30 ± 2% in 50 lM Cu medium when compared with APP+/+/APLP2–/– neurons (Fig. 1b). In APP–/–/APLP2–/– neurons, copper levels increased by 88 ± 3% in basal Cu medium, 102 ± 6% in 15.7 lM Cu medium and 59 ± 6% in 50 lM Cu medium when compared with APP+/+/APLP2–/– neurons (Fig. 1b). The increased copper accumulation observed between cortical neurons appeared to be related to a gene-dosage effect of APP expression when in an APLP2-deficient background. Copper



accumulation was significantly elevated in APP+/–/APLP2–/– neurons compared with APP+/+/APLP2–/– neurons, with greatest accumulation occurring in APP–/–/APLP2–/– neurons compared with APP+/+/APLP2–/– neurons (Fig. 1b). Increased copper accumulation for APP–/–/APLP2–/– mouse embryonic fibroblasts Mouse embryonic fibroblasts (MEFs) derived from wild-type (APP+/+/APLP2+/+), APP+/+/APLP2–/–, APP+/–/APLP2–/– and APP–/–/APLP2–/– mice were also investigated for differences in copper accumulation (Fig. 2). No significant difference in copper accumulation was observed between APP+/+/APLP2–/– and wild-type MEFs after both 24-h (Fig. 2a) and 48-h incubation periods (Fig. 2b). Furthermore, no significant difference in copper accumulation was

observed between APP+/+/APLP2–/–, APP+/–/APLP2–/– and wild-type MEFs (Fig. 2). However, increased copper accumulation was observed in double knockout APP–/–/APLP2–/– MEFs compared with wild-type MEFs (Fig. 2). This suggests that, in MEFs, the expression of APP in an APLP2–/– background is sufficient to maintain copper homeostasis. Over-expression of APP in Tg2576 primary cortical neurons decreases copper accumulation Tg2576 mice are transgenic for the Swedish mutation, APP695.K670N-M671L, and as such have increased b-secretase cleavage of APP (Hsiao et al. 1996). This results in increased secretion of both Ab and b-secretase cleaved APP (Hsiao et al. 1996). To determine the effect of overexpression of APP on neuronal copper accumulation primary mouse neurons derived from Tg2576, mice were investigated for copper accumulation. Significant decreases in copper accumulation were observed between Tg2576 neurons compared with NTg controls in basal (0.76 lM), 50, 100 and 150 lM Cu medium (Fig. 3a). Copper levels decreased by 26% from NTg control neurons compared with Tg2576 neurons in basal Cu medium, by 27% in 50 lM Cu medium, 18% in 100 lM Cu medium and 17% in 150 lM Cu medium (Fig. 3b). Discussion

Fig. 2 Effect of APP and APLP2 gene knockout on copper accumulation in mouse embryonic fibroblasts. Copper levels were measured, using 64Cu, in mouse embryonic fibroblasts (MEFs) derived from wildtype (APP+/+/APLP2+/+), APP+/+/APLP2–/–, APP+/–/APLP2–/– and APP–/–/APLP2–/– knockout mice. (a) MEF cultures were incubated in medium containing 64Cu and ‘no added copper’ (basal) or medium with added ‘cold’ CuCl2 to give a total copper concentration of 15.7, 32 and 50 lM for 24 h. Significant increases in copper accumulation were observed in double knockout (APP–/–/APLP2–/–) derived MEFs in 15.7, 32 and 50 lM copper-containing media. Values for copper accumulation are expressed as pmol Cu/mg protein. Bars represent the mean ± SE from three to five independent triplicate experiments (ANOVA, Bonferroni’s post test; ***p < 0.001 compared with APP+/+/ APLP2+/+ MEFs). (b) MEFs cultures were incubated in medium containing 64Cu and ‘no added copper’ (basal) or medium with added ‘cold’ CuCl2 to give a total copper concentration of 15.7, 32 and 50 lM for 48 h. Significant increases in copper accumulation were observed in double knockout (APP–/–/APLP2–/–) derived MEFs in basal, 15.7, 32 and 50 lM copper-containing media. Values for copper accumulation are expressed as pmol Cu/mg protein. Bars represent the mean ± SE from three independent triplicate experiments (ANOVA, Bonferroni’s post test; ***p < 0.001 compared with APP+/+/APLP2+/+ MEFs).

The present study utilizes cellular radio-copper assays to directly examine the role of APP and APLP2 proteins in modulating cellular copper homeostasis in neuronal and fibroblast cell lines. We demonstrate for the first time a gene dosage-dependent effect of APP expression on cellular copper levels for cultured primary mouse cortical neurons that have a genetic ablation of APLP2 expression. In contrast, we show that over-expression of APP in primary cortical neurons reduces cellular copper levels. We also suggest that APP and APLP2 can functionally substitute for each other in maintaining copper homeostasis in cultured primary neurons. Copper levels are increased in brain and liver of APP–/– and APLP2–/– mice, suggesting that both proteins function in modulating copper levels (White et al. 1999b). This observation is supported by the conserved N-terminal located copper-binding domain (Hesse et al. 1994; White et al. 2002; Barnham et al. 2003) and the proposed functional redundancy between APP and APLP2 family members (von Koch et al. 1997; Heber et al. 2000). Previously, we have demonstrated no difference in neuronal copper accumulation between APP–/– and wild-type neurons (White et al. 1999a). However, it is suggested that APLP2 may substitute for loss of APP function. APLP2 does not produce Ab, so its influence on copper levels is likely to be mediated by its N-terminal


APP and APLP2 function in neuronal copper homeostasis 427

Fig. 3 Effect of APP over-expression on copper accumulation in Tg2576 primary mouse cortical neurons. (a) Copper levels were measured in primary mouse cortical neurons derived from Tg2576 and NTg control mice. Primary cortical cultures were incubated in medium containing 64Cu and ‘no added copper’ (basal, 0.76 lM Cu) or medium with added ‘cold’ CuCl2 to give a total copper concentration of 50, 100 and 150 lM for 48 h. Significantly decreased copper accumulation was observed in Tg2576 neurons compared with NTg littermate control neurons. Values for copper accumulation are expressed as pmol Cu/mg protein. Bars represent the mean ± SE from four independent triplicate experiments (t-test; *p < 0.05; ***p < 0.001 compared with NTg neurons). (b) Percentage copper accumulation in Tg2576 neurons compared with NTg neurons. The mean copper accumulation from NTg control neurons was used to define 100% copper accumulation. Bars represent the mean ± SE from four independent triplicate experiments (t-test; *p < 0.05; ***p < 0.001 compared with NTg neurons).

copper-binding domain, which is homologous with that of APP (Hesse et al. 1994). Our results show that removal of APLP2 (APP+/+/APLP2–/–) expression had no effect on neuronal or MEF copper accumulation compared with wildtype (APP+/+/APLP2+/+) cultures (Figs 1 and 2). While this suggests that APLP2 has a minor role in copper homeostasis, like our previous findings using APP–/– cortical neurons (White et al. 1999a), it is possible that APP is substituting for the loss of function of APLP2. This notion was supported when heterozygote and homozygote knockout of APP expression in the APLP2–/– background resulted in significant increases in copper accumulation (Fig. 1). Heterozygote knockout of APP expression combined with homozygote knockout of APLP2 expression (APP+/–/APLP2–/–) significantly increased neuronal copper accumulation (Fig. 1), but had no effect on

copper accumulation in MEFs (Fig. 2). In both neuronal and MEFs cultures, homozygote knockout of APP and APLP2 expression (APP–/–/APLP2–/–) significantly increased copper accumulation (Figs 1 and 2). Furthermore, copper accumulation increased 2-fold in APP–/–/APLP2–/– neurons compared with APP+/–/APLP2–/– neurons, and agrees with the 50% reduction in APP expression in APP+/–/APLP2–/– (data not shown; Heber et al. 2000). There is no up-regulation of APP expression to compensate for the loss of APP family member function in brain homogenates and neuronal cultures (White et al. 1998; Heber et al. 2000). These results demonstrate a gene dosage-dependant effect of APP on neuronal copper levels. As no difference was observed in neuronal copper accumulation between APLP2–/– and wild-type neurons in this study (Fig. 1), and between APP–/– and wild-type neurons (White et al. 1999a), it suggests that APP and APLP2 are functionally interchangeable in modulating neuronal copper homeostasis. A likely mechanism is the secretion of the conserved APP and APLP2 N-terminal copper-binding domain (Hesse et al. 1994). The data utilising primary cultured neurons support this hypothesis. Over-expression of APP in Tg2567 primary neuronal cultures resulted in significantly decreased copper accumulation (Fig. 3). This observation supported the in vivo data that Tg2576 mice have reduced brain copper levels compared with age-matched controls (Maynard et al. 2002). The likely mechanism for the decreased copper accumulation in Tg2576 neurons is increased secretion of APP with the N-terminal copper-binding domain. This is hypothesised from: (i) Tg2576 mice have reduced brain copper levels prior to the appearance of Ab plaques (Hsiao et al. 1996; Maynard et al. 2002); (ii) the non-amyloidogenic processing of APP (Borchardt et al. 1999), and in vivo reduction of Ab levels in APP transgenic mice under elevated copper conditions (Bayer et al. 2003; Phinney et al. 2003). However, as overexpression of the CT100 fragment of APP, which lacks the N-terminal copper binding domain but expresses Ab, reduces brain copper levels in transgenic mice (Maynard et al. 2002), Ab may also be a factor in reducing copper levels in Tg2576 neurons. This current study has better defined the role of copperbinding proteins APP and APLP2 in neuronal copper homeostasis. Our results demonstrate a gene-dosage dependant effect of APP expression on neuronal cellular copper accumulation, with the likely mechanism modulated by the APP N-terminal copper-binding domain. Therefore, APP is a key modulator of neuronal copper homeostasis (Maynard et al. 2002; Barnham et al. 2003; Bayer et al. 2003; Bellingham et al. 2004). Moreover, neither knockout of APP nor APLP2 expression alone could influence neuronal copper levels, suggesting that APP and APLP2 have interchangeable functional roles in modulating neuronal copper homeostasis.



Acknowledgements This work was supported in part by grants from the Minerals Council of Australia, the Foundation of Young Australians, and Loxton Bequest (to SAB), National Health and Medical Research Council of Australia (to RC and CLM), and the Australian Institute of Nuclear Science and Engineering (to JC). We would like to thank Hui Zheng and Sam Sisodia for kindly providing APP and APLP2 knockout mice, respectively. We would also like to thank the Department of Pathology Animal House staff for their assistance and maintenance of mouse stocks.

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