Cytotoxic Effect of β‐Amyloid on a Human Differentiated Neuron Is Not Mediated by Cytoplasmic Ca2+ Accumulation

Abstract

The effects of synthetic beta-amyloid (A beta1-42) on cell viability and cellular Ca2+ homeostasis have been studied in the human neuron-like NT2N cell, which differentiates from a teratocarcinoma cell line, NTera2/C1.D1, by retinoic acid treatment. NT2N viability was measured using morphological criteria and fluorescent live/dead staining and quantified using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide metabolism. A beta1-42 dose-dependently caused NT2N cell death when it was present in the cell culture for 14 days but had no effect on viability when it was present for 4 days. The lowest effective concentration was 4 microM, and the strongest effect was produced by 40 microM. Control NT2N cells produced spontaneous cytosolic Ca2+ oscillations under basal conditions. These oscillations were inhibited dose-dependently (0.4-40 microM) by A beta1-42 that was present in the cell culture for 1 or 4 days. Ca2+ wave frequency was decreased from 0.21 +/- 0.02 to 0.05 +/- 0.02/min, amplitude from 88 +/- 8 to 13 +/- 4 nM, and average Ca2+ level from 130 +/- 8 to 58 +/- 3 nM. The Ca2+ responses to 30 mM K+ and 100 microM glutamate were not different between control and A beta-treated cells. Thus, the results do not support the hypothesis that cytosolic early Ca2+ accumulation mediates A beta-induced NT2N cell death.