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. 2009 May 1;284(18):12145-52.
doi: 10.1074/jbc.M808497200. Epub 2009 Mar 10.

Intracellular trafficking of presenilin 1 is regulated by beta-amyloid precursor protein and phospholipase D1

Yun Liu  1 , Yun-Wu Zhang, Xin Wang, Han Zhang, Xiaoqing You, Francesca-Fang Liao, Huaxi Xu
Affiliations

Intracellular trafficking of presenilin 1 is regulated by beta-amyloid precursor protein and phospholipase D1

Yun Liu et al. J Biol Chem. .

Abstract

Excessive accumulation of beta-amyloid peptides in the brain is a major cause for the pathogenesis of Alzheimer disease. beta-Amyloid is derived from beta-amyloid precursor protein (APP) through sequential cleavages by beta- and gamma-secretases, whose enzymatic activities are tightly controlled by subcellular localization. Delineation of how intracellular trafficking of these secretases and APP is regulated is important for understanding Alzheimer disease pathogenesis. Although APP trafficking is regulated by multiple factors including presenilin 1 (PS1), a major component of the gamma-secretase complex, and phospholipase D1 (PLD1), a phospholipid-modifying enzyme, regulation of intracellular trafficking of PS1/gamma-secretase and beta-secretase is less clear. Here we demonstrate that APP can reciprocally regulate PS1 trafficking; APP deficiency results in faster transport of PS1 from the trans-Golgi network to the cell surface and increased steady state levels of PS1 at the cell surface, which can be reversed by restoring APP levels. Restoration of APP in APP-deficient cells also reduces steady state levels of other gamma-secretase components (nicastrin, APH-1, and PEN-2) and the cleavage of Notch by PS1/gamma-secretase that is more highly correlated with cell surface levels of PS1 than with APP overexpression levels, supporting the notion that Notch is mainly cleaved at the cell surface. In contrast, intracellular trafficking of beta-secretase (BACE1) is not regulated by APP. Moreover, we find that PLD1 also regulates PS1 trafficking and that PLD1 overexpression promotes cell surface accumulation of PS1 in an APP-independent manner. Our results clearly elucidate a physiological function of APP in regulating protein trafficking and suggest that intracellular trafficking of PS1/gamma-secretase is regulated by multiple factors, including APP and PLD1.

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Figures

FIGURE 1.
FIGURE 1.
APP regulates steady state levels of PS1 at the cell surface. A, APP wild type (WT), APP/APLP2 double knock-out (dKO), and APP dKO cells transiently expressing either control pcDNA (pc) or APP were biotinylated. The cell lysates were incubated with streptavidin-agarose beads for affinity precipitation of biotinylated cell surface proteins. Biotinylated proteins and total cell lysates were subjected to SDS-PAGE and Western blot. B, HeLa cells (H), HeLa cells stably expressing APP Swedish mutations (H-sw), N2a (N), and N2a cells stably expressing APP695 (N-695) were biotinylated and affinity-precipitated, followed by SDS-PAGE and Western blot. C, mouse embryonic fibroblasts and N2a cells were transiently transfected with an APP small hairpin RNA construct to down-regulate APP or with a scrambled (sc) small hairpin RNA control construct. The cells were then subjected to biotinylation, and biotinylated cell surface proteins were precipitated with streptavidin-agarose beads for SDS-PAGE. D, APP WT, APP dKO, and APP dKO cells transiently transfected with pcDNA (pc), APP C99, C99 with a TGN retention signal (C99T), C99 with an ER retention signal (C99E), or APP lacking 57 amino acids at the carboxyl terminus (Δ57) were biotinylated. Affinity-precipitated cell surface proteins were analyzed by Western blot. Antibodies used for recognizing APP, PS1 NTF, and BACE1 were 369, Ab14, and B690, respectively. Protein levels were quantitated by densitometry and normalized to respective controls (set as 1 arbitrary unit). The data represent the means ± S.E. from three separate experiments. *, p < 0.05.
FIGURE 2.
FIGURE 2.
APP deficiency accelerates cell surface delivery of PS1. After labeling with [35S]methionine, APP WT and APP dKO cells were incubated at 20 °C to promote accumulation of isotope-labeled protein in the TGN. The cells were then switched to 37 °C for indicated times and biotinylated at 4 °C. The cell lysates were subjected to affinity precipitation with streptavidin-agarose beads and immunoprecipitation with a PS1 antibody (Ab14 for NTF), followed by SDS-PAGE and autoradiography. PS1 levels were normalized to those of PS1 at 5 min of chase in APP WT. The data represent the means ± S.E. from three experiments.
FIGURE 3.
FIGURE 3.
APP deficiency increases cell surface levels of PS1. A, APP WT and APP dKO cells were homogenized and fractionated by sucrose gradient sedimentation (left panels). One-ml samples from each cell line were collected from the top to the bottom of the gradient (labeled from 1 to 11). Equal sample volumes were analyzed by Western blot for PS1 NTF. Bip, γ-adaptin, Na/K ATPase, and Rab5 served as markers for ER, Golgi/TGN, PM, and vesicles, respectively. The level of PS1 NTF in each fraction was quantitated by densitometry. The ratio of PS1 NTF level in the ER/PM fractions relative to the total PS1 NTF level was analyzed and normalized to that of APP WT cells (set as 1 arbitrary unit). The data represent the means ± S.E. from three separate experiments. *, p < 0.05 (right panels). B, APP WT and dKO cells were fixed, permeabilized, sequentially incubated with anti-PS1 NTF antibody Ab14 and Alexa Fluor 488-conjugated secondary antibody, and examined with deconvolution microscope. The arrows indicate cell surface PS1.
FIGURE 4.
FIGURE 4.
APP regulates cell surface delivery of all four γ-secretase components and modulates Notch cleavage. A, APP dKO cells were transiently transfected with pcDNA control or APP cDNA and subjected to biotinylation. Affinity-precipitated cell surface proteins were analyzed by Western blot for PS1 NTF, NCT, PEN-2, and APH-1. Protein levels were quantitated by densitometry and normalized to respective controls (set as 1 arbitrary unit). The data represent the means ± S.E. from three experiments. *, p < 0.05. B, APP dKO cells were transiently transfected with a Notch NΔE-Myc vector. After splitting equally, the cells were transfected with pcDNA (pc) or different amounts of APP cDNA and biotinylated. The cell lysates were subjected to SDS-PAGE and Western blot with antibodies against total and biotinylated (surface) APP (369), PS1 (Ab14), and NΔE (using the 9E10 anti-Myc antibody) and cleaved NΔE/NICD. C, samples transfected with 8 μg of control (APP 0 μg) or APP cDNA in B were used for comparison. After densitometry quantitation, relative levels of NICD/total NΔE were normalized to those of controls (set as 1 arbitrary unit). The data represent the means ± S.E. from three experiments. *, p < 0.05.
FIGURE 5.
FIGURE 5.
PLD1 regulates PS1 trafficking. PS wild type cells (PS WT) and PS1/PS2 double knock-out (PS dKO) cells (A) and APP WT and APP dKO cells were transiently transfected with pcDNA (pc), wild type PLD1 (wt), or a catalytically inactive form of PLD1 (K898R, mut) (B). The cells were then subjected to biotinylation and analysis of cell surface proteins. C, APP dKO cells were first transfected with control vector (-) or APP (+). After splitting equally, the cells were transfected with wt or mut PLD1 followed by biotinylation to analyze cell surface proteins. Antibodies 369 and Ab14 recognizing APP and PS1 NTF, respectively, were used for Western blot analysis. Protein levels were quantitated by densitometry and normalized to respective controls (set as 1 arbitrary unit). The data represent the means ± S.E. from three experiments. *, p < 0.05.
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