Zinc Inhibits Amyloid beta Production from Alzheimer's Amyloid Precursor Protein in SH-SY5Y Cells

Abstract

Zinc released from excited glutamatergic neurons accelerates amyloid beta (Abeta) aggregation, underscoring the therapeutic potential of zinc chelation for the treatment of Alzheimer's disease (AD). Zinc can also alter Abeta concentration by affecting its degradation. In order to elucidate the possible role of zinc influx in secretase-processed Abeta production, SH-SY5Y cells stably expressing amyloid precursor protein (APP) were treated with pyrrolidine dithiocarbamate (PDTC), a zinc ionophore, and the resultant changes in APP processing were examined. PDTC decreased Abeta40 and Abeta42 concentrations in culture media bathing APP-expressing SH-SY5Y cells. Measuring the levels of a series of C-terminal APP fragments generated by enzymatic cutting at different APP-cleavage sites showed that both beta- and alpha-cleavage of APP were inhibited by zinc influx. PDTC also interfered with the maturation of APP. PDTC, however, paradoxically increased the intracellular levels of Abeta40. These results indicate that inhibition of secretase-mediated APP cleavage accounts -at least in part- for zinc inhibition of Abeta secretion.

Keywords: Amyloid beta; Amyloid precursor protein; Pyrrolidine dithiocarbamate; Zinc.

Fig. 1

PDTC decreases Aβ levels in the conditioned media. PDTC was added to SH-SY5Y-wt cells at the concentration indicated for 4 h. Aβ40 (A) and Aβ42 (B) in the conditioned media were quantified by sandwich ELISA. Bars represent the means±S.D. of three independent experiments. Control represents no drug treatment. Statistical significance was represented as ^*(p<0.05).

Fig. 2

Zinc is required for PDTC action. Aβ40 levels in the conditioned media from SH-SY5Y-wt cells were measured by sandwich ELISA (A and B) or by TCA precipitation followed by combined immunoprecipitation and immunoblot assays with Aβ40 antibody (C). (A) SH-SY5Y-wt cells were incubated with or without 100-µM PDTC and/or 10-µM EDTA for 4 h. (B) Serum-deprived SH-SY5Y-wt cells were treated as follows: control, 100-µM PDTC, 100-µM PDTC with 1.5-µM ZnSO₄ or 1.5-µM CuSO₄, 1.5-µM ZnSO₄, and 150-µM ZnSO₄ for 4 h. (C) The serum-free conditioned media from SH-SY5Y-wt cells treated with or without PDTC plus 1.5-µM zinc were concentrated by TCA precipitation for Aβ40 assay (see methods). Bars represent the mean±S.D. of three different experiments. Control represents no drug treatment. Statistical significance was represented as ^*(p<0.05).

Fig. 3

APP processing by β- and α-secretase is inhibited by PDTC. The cell lysates (A) or conditioned media (B) of SH-SY5Y-wt cells treated with or without 100-µM PDTC were analyzed by immunoblot assay (B) or combined immunoprecipitation-immunoblot assay (A) (see methods). The amounts of C99 and C83 detected in immunoblot assays with APP C-terminus antibodies were semi-quantified with densitometry. A monoclonal 6E10 antibody (against a.a. 1~17 of human Aβ) was used for the sAPPα assay. Bars represent the means±S.D. of two or three independent experiments. Control represents no drug treatment. Mature APP is represented as mAPP; immature APP, imAPP. Statistical significance was represented as ^*(p<0.05).

Fig. 4

PDTC increases intracellular Aβ40. SH-SY5Y-swe cells were incubated with or without 100-µM PDTC for 4 h and then scraped, pelleted and lysed. Aβ40 in the cell lysates was assayed by sandwich ELISA. Bars represent the mean±S.D. of three independent experiments. Control represents no drug treatment. Statistical significance was represented as ^*(p<0.05).

Fig. 5

Maturation of APP is inhibited by PDTC. Cell lysates of SH-SY5Y-wt cells treated with or without 100-µM PDTC for 4 h were separated in 7% SDS-PAGE. (A) APP was immunoblotted with APP C-terminus antibody. (B) Quantitative analysis of the holoAPP bands (mature form+immature form). Amount of holoAPP was normalized to actin levels. (C) The maturation degree of APP was shown by the percentage of mAPP to imAPP. Bars represent the mean±S.D. of three independent experiments. Control represents no drug treatment. Statistical significance was represented as ^*(p<0.05).