Abstract
Familial Alzheimer’s disease (AD) is caused by mutations in the genes that encode amyloid precursor protein (APP) and presenilins. Disturbances in calcium homeostasis have been observed in various cellular and animal models of AD and are proposed to underlie the pathogenesis of the disease. Furthermore, wildtype presenilins were shown to regulate endoplasmic reticulum (ER) calcium homeostasis, although their precise mechanism of action remains controversial. To investigate whether APP also affects ER calcium levels, we used RNA interference to target the APP gene in cultured T84 cells in combination with two types of ER calcium sensors. Using a genetically encoded calcium indicator, GEM-CEPIA1er, we found that APP-deficient cells exhibited elevated resting calcium levels in the ER and prolonged emptying of ER calcium stores upon the cyclopiazonic acid-induced inhibition of sarco-endoplasmic reticulum calcium-ATPase. These effects could be ascribed to lower ER calcium leakage rates. Consistent with these results, translocation of the endogenous ER calcium sensor STIM1 to its target channel Orai1 was delayed following ER calcium store depletion. Our data suggest a physiological function of APP in the regulation of ER calcium levels.
Highlights
Calcium (Ca2+) is a versatile cellular second messenger[1]
The specificity of the antibodies was confirmed in T84 cells, in which ORAI1 and stromal interaction molecule 1 (STIM1) expression was knocked down by RNA interference (RNAi) using virally-delivered specific short-hairpin RNA sequences (Fig. 1b,c)
As demonstrated in this work using GEM-CEPIA1er, T84 cells with downregulated amyloid precursor protein (APP) expression had substantially elevated resting [Ca2+]endoplasmic reticulum (ER) compared with APP-containing cells
Summary
Calcium (Ca2+) is a versatile cellular second messenger[1]. It plays an important role in a multitude of cellular activities, ranging from gene transcription to neurotransmission. The Ca2+-related functions of APP-derived fragments were inferred solely from changes in cytosolic Ca2+ levels Using both cytosolic and ER-targeted Ca2+ indicators, Oules et al recently reported that the overexpression of a FAD-causing APPSWE mutant reduced ER Ca2+ load capacity through higher activity of Ca2+ release mechanisms from this organelle[28]. We used a different approach to shed light on the physiological function of APP and examined ER Ca2+ levels in cells with downregulated APP expression For this purpose, we used both the ER-targeted genetically encoded Ca2+ indicator (GECI) GEM-CEPIA1er[29] and the endogenous ER Ca2+ sensor STIM1. Our data suggest a regulatory role for APP in ER Ca2+ homeostasis
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