A physiologic signaling role for the gamma -secretase-derived intracellular fragment of APP

Abstract

Presenilins mediate an unusual intramembranous proteolytic activity known as gamma-secretase, two substrates of which are the Notch receptor (Notch) and the beta-amyloid precursor protein (APP). Gamma-secretase-mediated cleavage of APP, like that of Notch, yields an intracellular fragment [APP intracellular domain (AICD)] that forms a transcriptively active complex. We now demonstrate a functional role for AICD in regulating phosphoinositide-mediated calcium signaling. Genetic ablation of the presenilins or pharmacological inhibition of gamma-secretase activity (and thereby AICD production) attenuated calcium signaling in a dose-dependent and reversible manner through a mechanism involving the modulation of endoplasmic reticulum calcium stores. Cells lacking APP (and hence AICD) exhibited similar calcium signaling deficits, and-notably-these disturbances could be reversed by transfection with APP constructs containing an intact AICD, but not by constructs lacking this domain. Our findings indicate that the AICD regulates phosphoinositide-mediated calcium signaling through a gamma-secretase-dependent signaling pathway, suggesting that the intramembranous proteolysis of APP may play a signaling role analogous to that of Notch.

Figure 1

Fibroblasts from presenilin knockout mice show impairments in calcium signaling that correlate with Aβ production. (a) Representative calcium signals evoked by BK (100 nM) in fibroblasts from mice lacking both presenilin genes or controls. (b) Comparison of calcium signal amplitude (dark bars) and [Aβ_1–40] in the conditioned medium (light bars) of fibroblasts from presenilin knockout mice. (Inset) The deficits in calcium signaling in PS1^−/− mice are completely reversed by transfection with wild-type PS1. Calcium data are mean ± SE for 8–24 experiments per condition. ELISA data are mean ± SE of triplicate determinations. *, P < 0 01.

Figure 2

Pharmacological inhibition of γ-secretase impairs calcium signaling. (a) Typical BK-evoked calcium signals in N2aWt11 cells treated with various concentrations of MW-III-36C. (b) Effects of a representative γ-secretase inhibitor on both BK-evoked calcium signal amplitude (open circles) and [Aβ_1–40] in the conditioned medium (closed circles) after incubation in various concentrations of compound for 3–4 h. Calcium data are mean of quadruplicate determinations. ELISA data are mean of triplicate determinations. (c) The calcium deficits produced by γ-secretase inhibitors are reversible (see text for details). Data are mean ± SE of 6 experiments per condition. (d) Time course of effectiveness of IL on calcium signal amplitude (open circles) and [Aβ_1–40] in the conditioned medium (closed circles). Calcium data are mean ± SE of 4 experiments per condition; ELISA data are mean of triplicate determinations *, P < 0.01.

Figure 3

APP^−/− fibroblasts exhibit deficits in calcium signaling resembling PS1^−/− cells. (a) Representative BK-evoked calcium signals from APP^−/− fibroblasts and controls. The amplitude of calcium signals is diminished in APP^−/− cells (b) but increased in Chinese hamster ovary cells overexpressing APP (c). Data are mean ± SE of 6 experiments per condition. *, P < 0.01.

Figure 4

The calcium deficits in APP^−/− cells can be rescued by transfection with constructs containing the AICD. (a) Schematic illustration of the APP constructs used for transfections. (b) Rescue of the calcium impairments in APP^−/− cells was achieved after transfection with constructs containing an intact AICD or AICD alone, but not by constructs lacking this domain. Data are mean ± SE of 8 experiments per condition. *, P < 0.05 relative to APP^−/− cells.

Figure 5

Cells with impaired AICD production exhibit reduced ER calcium levels. To quantify ER calcium content, calcium stores were depleted with 1 μM thapsigargin in calcium-free medium. (a) Representative traces from control and PS1^−/−/PS2^−/− cells. (b–d) Amplitude of thapsigargin-evoked calcium transients in (b) PS1^−/−/PS2^−/− cells, (c) APP^−/− cells, and (d) in cells treated with various γ-secretase inhibitors (or PIA, an inactive control). Data are mean + SE of 4–6 experiments per condition. *, P < 0.01.