Amyloid precursor protein (APP) and copper homeostasis in the human neuroblastoma cell line SH-SY5Y

Abstract

Alzheimer’s disease (AD) is characterised by cerebellar accumulation and aggregation of amyloid beta (Aβ), a cleavage product of the transmembrane amyloid precursor protein (APP). APP contains a range of functional domains in its large extracellular portion, among which are two copper-binding motifs and one zinc-binding motif. The copper-binding motifs are present in the amino-terminal region of APP and within the Aβ region of the protein and readily reduce Cu(II) to Cu(I), thus APP and its cleavage products are linked to copper metabolism and have been hypothesised to participate in cellular copper homeostasis. In this project human neuroblastoma cell lines SH-SY5Y were utilised to determine the effect of expressing a familial AD mutation on intracellular copper concentrations and possible functional alterations or deficits of enzymes that require copper as a co-factor. The familial AD mutation first found in a Swedish population was previously shown to increase the total amount of released Aβ. Direct phenotypic comparison between SH-SY5Y APPWT cell lines expressing endogenous levels of APP and APPswe Coupling native two dimensional liquid chromatography with metal analysis, SDS-PAGE and Principal Component Analysis identified one major copper and zinc containing pool as copper-zinc superoxide dismutase (SOD1) in soluble whole cell protein extracts. Comparative analysis of metal content between APP cell lines overexpressing APP carrying the Swedish mutation was performed in standard culture and manipulated copper concentrations. WT and APPswe cultures indicated a difference in metallation of SOD1 with copper. APPswe cultures displayed reduced metallation of SOD1, whereas SOD1 metallation with zinc remained unaltered. Functional analysis of copper-binding enzymes, such as SOD1 and cytochrome c oxidase (CCO), using standard biochemical approaches, identified lower activities for both enzymes during standard cell culture in APPswe cells. Upon treatment of cultures with increasing concentrations of exogenous copper APPWT enzyme activities remained unaltered but enzyme activities in APPswe cultures increased in direct correlation with increasing copper concentrations. Combined with phenotypic analysis of growth, survival and intracellular metal content it appears that APPswe cultures are copper deficient, but this can be overcome by copper supplementation. Lower copper accumulation also enables greater survival of APP swe Overall, these data suggest that APP cells in elevated copper. swe overexpression in SH-SY5Y cells results in functional copper deficiency which can be rescued by supplementation of cultures with exogenous copper. APPswe further confers resistance to copper toxicity not only via increased Aβ release, but also via increased copper delivery to enzymatic target proteins improving cellular antioxidant response and energy metabolism. These data are consistent with a function of APP in copper efflux either in a regulatory capacity or directly contributing to copper egress.