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.
Highlights
Imbalance of metal ions has been recognized as one of the key factors in the pathogenesis of Alzheimer disease (AD).2 Aberrant interactions between copper or zinc with the -amyloid peptide (A) released into the glutamatergic synaptic cleft vicinity could result in the formation of toxic A oligomers and aggregation into plaques characteristic of AD brains
Growing evidence suggests that amyloidogenic processing of the -amyloid precursor protein (APP) occurs in lipid rafts, membrane microdomains enriched in cholesterol. - and ␥-secretases, and A 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
Copper and cholesterol have been reported independently in the bution to lipid raft fractions was significantly greater in these literature as modulating factors of APP processing and A genbrain tissues (Fig. 5b)
Summary
Cell Culture—SH-SY5Y human neuroblastoma cells were cultured in RPMI media 1640 with GLUTAMAXTM-I (Invitrogen) supplemented with 20% fetal calf serum. After metal treatments as described above, cells were resuspended in 0.5 ml of lysis buffer containing 25 mM Tris-HCl (pH 7.4), 150 mM NaCl, 5 mM EDTA, and 0.5% Brij-58 (Sigma) supplemented with CompleteTM EDTA-free protease inhibitor mixture (Roche). For ␥-secretase activity assay, sucrose gradient fractions, diluted with lysis buffer, were concentrated by ultracentrifugation at 55,000 ϫ g at 4 °C for 70 min in a Beckman TLA55 rotor. Metal Analysis—SH-SY5Y cells and mice brain homogenates were pre-digested overnight in 50 l of concentrated HNO3 (Aristar Grade, BDH), followed by heating to 90 °C for 20 min. The digested homogenate and sucrose gradient fraction samples were diluted with 1% HNO3 and metal levels (copper, iron, and zinc) were measured by inductively coupled plasma mass spectrometry (Ultramass 700, Varian, Australia). Significance was defined as *, p Յ 0.05; **, p Յ 0.01; ***, p Յ 0.001
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