Presenilin-1 uses phospholipase D1 as a negative regulator of beta-amyloid formation

Abstract

Presenilin (PS1/PS2) is a major component of gamma-secretase, the activity that mediates proteolysis of beta-amyloid precursor protein to generate beta-amyloid (Abeta). Here we demonstrate that PS1, through its loop region, binds to phospholipase D1 (PLD1), thereby recruiting it to the Golgi/trans-Golgi network. Overexpression of wild-type PLD1 reduces Abeta generation. Conversely, down-regulation of endogenous PLD1 by small hairpin RNA elevates Abeta production. The Abeta-lowering effect of PLD1 is independent of its ability to promote vesicular budding of beta-amyloid precursor protein. The data indicate that overexpression of PLD1 decreases, and down-regulation of PLD1 increases, the catalytic activity, and the association of the subunits, of gamma-secretase.

Fig. 1.

PS1, through its loop region, interacts with and recruits PLD1 to the Golgi/TGN. (a) Protein lysates from ES cells derived from PS1/PS2wt and PS1^−/−/PS2^−/− mice were immunoprecipitated with anti-PLD1 antibody followed by immunoblotting with anti-PS1 loop antibody, or vice versa. Ten percent of total protein lysate was loaded as input. Alternatively, ES cells overexpressing HA-tagged PLD2 were lysed, and protein lysates were immunoprecipitated with HA antibody followed by immunoblotting with anti-PS1 loop antibody. (b) The interaction between PS1 and PLD1 was assayed in a cell-free system derived from ES cells (PS1/PS2wt) with added anti-PS1 NTF antibody (epitope in blue), anti-PS1 loop antibody (epitope in red), or anti-rabbit IgG antibody used as a control. After incubation, the cell-free system was diluted into IP buffer and immunoprecipitated by anti-PLD1 antibody followed by immunoblotting with anti-PS1 antibody. Graph shows mean ± SE of three experiments. ∗∗, P < 0.001; Student’s t test. (c) Fibroblasts derived from PS1^+/+ and PS1^−/− mice were immunolabeled for endogenous PLD1 by antibody P1-P4 followed by rhodamine-conjugated secondary antibody (red fluorescence). (Insets) Enlargement of typical cells. γ-adaptin was used as a marker for Golgi apparatus and TGN (green fluorescence). Overlays represent digitally merged images. Yellow fluorescence indicates colocalization of PLD1 with γ-adaptin. (Scale bar, 10 μm.)

Fig. 2.

PLD1 inhibits Aβ production. (a) N2a cells expressing PS1ΔE9 were transiently transfected with PLD1wt or mock cDNA. Secreted Aβ, intracellular Aβ, full-length βAPP, sβAPPα, and βCTF levels were compared. ∗, P < 0.01; ∗∗, P < 0.001; Student’s t test. (b) Surface-enhanced laser desorption/ionization mass spectrographic analysis comparing Aβ_1–38, Aβ_1–40, and Aβ_1–42 secreted from ΔE9 cells transfected with PLD1wt or mock cDNA. (c) N2a cells expressing PS1ΔE9 were transiently transfected with control shRNA or PLD1 shRNA. Secreted Aβ, intracellular Aβ, full-length βAPP, sβAPPα, and βCTF levels were compared. ∗∗, P < 0.001; Student’s t test.

Fig. 3.

PLD1 inhibits γ-secretase activity via a mechanism distinct from its effect on βAPP trafficking. (a) N2aΔE9 cells were transiently transfected with PLD1 mutant K898R or mock cDNA, and secreted and intracellular Aβ levels were compared. ∗, P < 0.01; ∗∗, P < 0.001; Student’s t test. (b) ES PS1/PS2 wt cells were transiently transfected with HA-tagged PLD1wt or K898R cDNA. The interaction between PS1 and PLD1 was assayed by IP with anti-HA antibody followed by immunoblotting with anti-PS1 loop antibody. (c) N2aΔE9 cells overexpressing myc-tagged mNotchΔE (mNΔE) were cotransfected with either PLD1 cDNA or mock cDNA, followed by pulse labeling with [³⁵S]methionine at 37°C for 30 min, and were chased for 15–90 min. mNotchΔE and its cleavage product, NICD, were immunoprecipitated with anti-myc antibody followed by SDS/PAGE and autoradiography. ∗∗, P < 0.001; Student’s t test. The graph shows quantification of NICD generation at 90 min of incubation. (d) Cells were permeabilized and incubated at 37°C for 60 min to allow the formation of post-TGN vesicles. mNotchΔE in nascent vesicles was immunoprecipitated by anti-myc antibody followed by SDS/PAGE and autoradiography. Results are expressed as percentage of controls.

Fig. 4.

PLD1 disrupts the association of γ-secretase components. (a) Protein lysates from PS1/PS2 wt ES cells transfected with PLD1wt, PLD1 K898R, or mock cDNA were immunoprecipitated with anti-PEN2 antibody. The precipitates were subjected to SDS/PAGE and immunoblotting with anti-PS1 loop antibody (for PS1 CTF), Ab14 (for PS1 NTF), Ab716 [for nicastrin (Nct)], or anti-APH1 antibody. Ten percent of total protein lysates was loaded and shown as input lanes. Graphs show means ± SE of three experiments. ∗, P < 0.01; ∗∗, P < 0.001; Student’s t test. (b) Protein lysates from PS1/PS2 wt ES cells transfected with PLD1 shRNA or control shRNA were immunoprecipitated with anti-PEN2 antibody. The precipitates were subjected to SDS/PAGE and immunoblotting as described for a. Graphs show means ± SE of three experiments. ∗, P < 0.01; ∗∗, P < 0.001; Student’s t test. (c) Protein lysates from PS1^wt and PS1^−/− ES cells were immunoprecipitated with anti-PLD1 antibody and immunoblotted with anti-PEN2 antibody, or vice versa.

Fig. 5.

Model of the mechanisms by which PLD1 may antagonize PS1 regulation of βAPP metabolism and stimulate βAPP trafficking from the TGN. PS1 is required for the recruitment of PLD1 to the Golgi/TGN membranes (red arrowhead). The recruited PLD1 serves, in two ways, as a negative regulator of PS1 function. First, PS1 inhibits budding of βAPP-containing vesicles from the TGN, whereas PLD1 stimulates this process through a mechanism that is at least partially independent of PS1. Second, PLD1, through a direct interaction with PS1 and disruption of association of the γ-secretase subunits, inhibits the ability of the protease to cleave βCTF to generate Aβ. The present data do not exclude the possibility (i) that PLD1 might also antagonize PS1 inhibition of βAPP trafficking by its ability to bind directly to PS1, or (ii) that PLD1 might also inhibit Aβ generation through an action independent of its inhibitory effect on PS1 (dashed arrows). This scheme is based on the present study and that of Cai et al. (22).