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. 2009 Aug 14;284(33):21899-21907.
doi: 10.1074/jbc.M109.019521. Epub 2009 Jun 19.

Paradoxical condensation of copper with elevated beta-amyloid in lipid rafts under cellular copper deficiency conditions: implications for Alzheimer disease

Ya Hui Hung  1 , Elysia L Robb  2 , Irene Volitakis  3 , Michael Ho  4 , Genevieve Evin  5 , Qiao-Xin Li  4 , Janetta G Culvenor  6 , Colin L Masters  5 , Robert A Cherny  2 , Ashley I Bush  3
Affiliations

Paradoxical condensation of copper with elevated beta-amyloid in lipid rafts under cellular copper deficiency conditions: implications for Alzheimer disease

Ya Hui Hung et al. J Biol Chem. .

Abstract

Redox-active copper is implicated in the pathogenesis of Alzheimer disease (AD), beta-amyloid peptide (Abeta) aggregation, and amyloid formation. Abeta.copper complexes have been identified in AD and catalytically oxidize cholesterol and lipid to generate H2O2 and lipid peroxides. The site and mechanism of this abnormality is not known. Growing evidence suggests that amyloidogenic processing of the beta-amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. beta- and gamma-secretases, and Abeta have been identified in lipid rafts in cultured cells, human and rodent brains, but the role of copper in lipid raft amyloidogenic processing is presently unknown. In this study, we found that copper modulates flotillin-2 association with cholesterol-rich lipid raft domains, and consequently Abeta synthesis is attenuated via copper-mediated inhibition of APP endocytosis. We also found that total cellular copper is associated inversely with lipid raft copper levels, so that under intracellular copper deficiency conditions, Abeta.copper complexes are more likely to form. This explains the paradoxical hypermetallation of Abeta with copper under tissue copper deficiency conditions in AD.

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Figures

FIGURE 1.
FIGURE 1.
Copper induces flotillin-2 redistribution from lipid rafts. Lipid rafts were isolated from SH-SY5Y cells exposed for 3 h to differential copper conditions, or from brain tissues of copper-deficient 15-month-old female Tg2576 (n = 4) and age-matched non-Tg control (n = 3) mice on a discontinuous sucrose gradient by ultracentrifugation as described under “Experimental Procedures.” Lipid raft fractions were identified by Western blot analysis as flotillin-2 positive (a) fractions 3–5 in SH-SY5Y untreated control, and (c) fraction 5 in non-Tg mice brains. Cholesterol analysis further confirmed these flotillin-2-positive lipid raft fractions by their concentrated cholesterol content (b and d). D, copper-deficient (50 μm BCS and 50 μm d-penicillamine). Densitometry analysis of flotillin-2 levels in the indicated fractions were expressed as a percentage of total cellular or brain tissue flotillin-2. Cholesterol data were expressed as a percentage of total cellular or brain tissue cholesterol. Data represent the results of at least three independent experiments (mean ± S.E.). *, p ≤ 0.05; ***, p ≤ 0.001 compared with untreated SH-SY5Y cells or non-Tg control.
FIGURE 2.
FIGURE 2.
Copper exposure reduced Aβ levels within lipid rafts. Western blot analysis of (a) full-length APP in lipid rafts isolated from SH-SY5Y cells and (b) Aβ in lipid rafts isolated from SH-SY5Y cells overexpressing swAPP695, exposed to differential copper conditions for 3 h. c, Western blot analysis of Aβ in lipid rafts isolated from brain tissues of copper-deficient 15-month-old female Tg2576 (n = 4) and age-matched non-Tg control (n = 3) mice. APP and Aβ were detected using W0-2 antibody. Aβ was not detected in brain tissues of non-Tg control mice. D, copper-deficient (50 μm BCS and 50 μm d-penicillamine). Densitometry analyses of APP and Aβ in lipid raft fractions were expressed as a percentage of total cellular APP and Aβ, respectively. Cell data represent the results from at least three independent experiments (mean ± S.E.). *, p ≤ 0.05; **, p ≤ 0.01 compared with untreated SH-SY5Y cells or non-Tg control.
FIGURE 3.
FIGURE 3.
Copper alters distribution of γ-secretase to lipid rafts. a, γ-secretase activity in lipid rafts isolated from SH-SY5Y cells exposed to differential copper conditions for 3 h was determined by the release of ϵ-CTF from MC99–3FLAG peptide. γ-Secretase activity in lipid rafts was expressed as a percentage of the untreated control. Presenilin 1 N-terminal fragment (PS1 NTF) in lipid rafts isolated from (b) SH-SY5Y cells exposed to differential copper conditions for 3 h, and (c) brain tissues of 15-month-old female Tg2576 (n = 4) and age-matched non-Tg control (n = 3) mice was detected using an anti-PS1 N terminus (98/1) antibody by Western blotting. Nct in lipid rafts isolated from (d) SH-SY5Y cells exposed to differential copper conditions for 3 h, and (e) mouse brain tissue was detected using an anti-nicastrin antibody by Western blotting. The mature Nct was the predominant species present in lipid rafts. D, copper-deficient (50 μm BCS and 50 μm d-penicillamine). Densitometry analyses of PS1 NTF and Nct levels in lipid raft fractions were expressed as a percentage of total cellular or brain tissue PS1 and Nct levels, respectively. SH-SY5Y cell data represent the results from at least three independent experiments (mean ± S.E.). **, p ≤ 0.01; ***, p ≤ 0.001 compared with untreated SH-SY5Y cells or non-Tg control.
FIGURE 4.
FIGURE 4.
Copper promotes retention of APP at cell surface. Surface proteins biotinylated using sulfo-NHS-SS-biotin post-copper treatments were precipitated with NeutrAvidin-agarose, and analyzed by Western blotting. APP was detected using W0-2 antibody. Total APP were normalized to β-actin as a loading control, and expressed as a percentage of the untreated control. The ratio of surface APP to normalized total APP was expressed as a percentage of the untreated control. Data represent the results of two independent experiments (mean ± S.E.). *, p ≤ 0.05 compared with untreated SH-SY5Y cells.
FIGURE 5.
FIGURE 5.
Copper distribution to lipid rafts associates inversely with total cellular copper levels. Analysis of copper levels in lipid rafts isolated from (a) SH-SY5Y cells exposed to differential metal conditions for 3 h, and from (b) brain tissues of copper-deficient 15-month-old female Tg2576 (n = 4) and age-matched non-Tg control (n = 3) mice. Lipid raft copper levels were expressed as a percentage of total cellular or brain tissue copper (mean ± S.E.). Molar ratio of total cellular copper:cholesterol in (c) SH-SY5Y cells exposed to differential copper conditions, and (d) mouse brain tissues. Copper:cholesterol molar ratio in lipid rafts isolated from (e) SH-SY5Y cells exposed to differential copper conditions, and (f) mouse brain tissues. Data represent the results of at least three independent experiments (mean ± S.E.). D, copper-deficient (50 μm BCS and 50 μm d-penicillamine). *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 compared with untreated SH-SY5Y cells or non-Tg control.
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