Dysregulation of Ca²⁺ signaling in astrocytes from mice lacking amyloid precursor protein

Abstract

The relationship between altered metabolism of the amyloid-β precursor protein (APP) and Alzheimer's disease is well established but the physiological roles of APP still remain unclear. Here, we studied Ca(2+) signaling in primary cultured and freshly dissociated cortical astrocytes from APP knockout (KO) mice and from Tg5469 mice overproducing by five- to sixfold wild-type APP. Resting cytosolic Ca(2+) (measured with fura-2) was not altered in cultured astrocytes from APP KO mice. The stored Ca(2+) evaluated by measuring peak amplitude of cyclopiazonic acid [CPA, endoplasmic reticulum (ER) Ca(2+) ATPase inhibitor]-induced Ca(2+) transients in Ca(2+)-free medium was significantly smaller in APP KO astrocytes than in wild-type cells. Store-operated Ca(2+) entry (SOCE) activated by ER Ca(2+) store depletion with CPA was also greatly reduced in APP KO astrocytes. This reflected a downregulated expression in APP KO astrocytes of TRPC1 (C-type transient receptor potential) and Orai1 proteins, essential components of store-operated channels (SOCs). Indeed, silencer RNA (siRNA) knockdown of Orai1 protein expression in wild-type astrocytes significantly attenuated SOCE. SOCE was also essentially reduced in freshly dissociated APP KO astrocytes. Importantly, knockdown of APP with siRNA in cultured wild-type astrocytes markedly attenuated ATP- and CPA-induced ER Ca(2+) release and extracellular Ca(2+) influx. The latter correlated with downregulation of TRPC1. Overproduction of APP in Tg5469 mice did not alter, however, the stored Ca(2+) level, SOCE, and expression of TRPC1/4/5 in cultured astrocytes from these mice. The data demonstrate that the functional role of APP in astrocytes involves the regulation of TRPC1/Orai1-encoded SOCs critical for Ca(2+) signaling.

Astroglial calcium signalling in Alzheimer's disease

“…In hippocampal astrocytes isolated and cultured from neonatal 3xTg-AD mice, the amplitude of ATP-induced [Ca 2+ ] i transients as well as the store-operated Ca 2+ entry were significantly increased [125,137]. In contrast, store-operated Ca 2+ entry in astrocytes from APP-over-expressing Tg5469 AD mice was not affected, although the deletion of APP inhibited SOCE, possibly due to down-regulation of Orai1 and TRPC1 channels [142].…”

Histidine triad nucleotide-binding protein 1 (HINT1) regulates Ca2+ signaling in mouse fibroblasts and neuronal cells via store-operated Ca2+ entry pathway

“…[Ca 2ϩ ]cyt was measured with fura-2 using digital imaging (25). Mouse embryonic fibroblasts or Neuro 2A cells were loaded with fura-2 by incubation for 35 min in culture medium containing 3.3 M fura-2 AM (20 -22°C, 5% CO2-95% air).…”

“…Image acquisition and analysis were performed with a MetaFluor/MetaMorph Imaging System (Molecular Devices, Downingtown, PA). [Ca 2ϩ ]cyt was calculated by determining the ratio of fura-2 fluorescence emission (510 nm) excited at 380 and 360 nm, as previously described (25). Intracellular fura-2 was calibrated in situ separately in cultured mouse embryonic fibroblasts and Neuro 2A cells (25).…”

“…Membrane proteins were solubilized in SDS buffer containing 5% 2-mercaptoethanol and were separated by SDS-PAGE as previously described (25). The following antibodies were used: rabbit polyclonal anti-HINT1 (26); rabbit polyclonal anti-Orai1 (ProSci, Poway, CA); rabbit polyclonal anti-STIM1 (a gift from Dr. J. Roos, Torrey Pines Therapeutics, La Jolla, CA).…”

Store-Operated Ca2+ Entry

“…Altered Ca 2+ homeostasis has been also linked to neuronal damage caused by brain aging or Alzheimer's disease. A recent study suggested that the physiological role of amyloid-b precursor protein (APP), whose altered metabolism has been associated with Alzheimer's disease, is the regulation of TRPC1/Orai1-mediated SOCE which is critical for Ca 2+ signaling in astrocytes [ 253 ] . In line with this report, impaired SOCE was also observed in fi broblasts bearing mutated presenilin-1 and 2 genes, which has been associated to autosomal dominant familial Alzheimer's disease (FAD), while absence of this protein led to increased SOCE and augmented expression of STIM1 [254][255][256][257] .…”