The APP670/671 mutation alters calcium signaling and response to hyperosmotic stress in rat primary hippocampal neurons

Abstract

Altered calcium homeostasis is implicated in the pathogenesis of Alzheimer's disease and much effort has been put into understanding the association between the autosomal dominant gene mutations causative of this devastating disease and perturbed calcium signaling. We have focused our attention on the effect of the APP670/671 mutation on spontaneous calcium oscillations in embryonic hippocampal neurons derived from the tg6590 transgenic rat. Intracellular free calcium levels were imaged by confocal microscopy using the fluorescent dye fluo-3AM. Hyperosmotic shrinkage, which can occur in a variety of pathophysiological conditions, has been shown to induce multiple cellular responses, including activation of volume-regulatory ion transport, cytoskeletal reorganization, and cell death. When exposed to hyperosmotic stress (addition of 50 mM sucrose) the frequency of calcium oscillations was suppressed to an equal extent in both wild-type and transgenic cultures, but the transgenic neurons, in contrast to the wild-type neurons, responded with a significantly higher increase in the amplitude of oscillations. A decrease in cell viability was observed by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay in neurons exposed to the hyperosmotic medium for 30 h. Whereas this loss in cell viability was comparable in both sets of cultures, the amplitude of calcium oscillations in transgenic neurons exhibited a significantly greater decrease in the presence of the L-type calcium channel antagonist, nimodipine. These results suggest that APP670/671 transgenic neurons have impaired calcium homeostasis.