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PLD3 gene and processing of APP

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Figure 1: Genetic deletion of Pld3 does not alter APP expression and processing in vivo.
Figure 2: Loss-of-function of Pld3 does not affect amyloid plaques burden in Appki mice.


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Authors and Affiliations



P.F. conceived the project, planned the experiments, performed the experiments, analysed the results and wrote the manuscript; K.H. performed in vitro experiments, and performed western blot and ELISA analysis in vitro and in vivo (Fig. 1 and Extended Data Fig. 2); A.M.A. performed electron microscopy experiments and helped to analyse the data; C.S.F. performed and analysed qPCR experiments (Extended Data Fig. 1b, c); T.S. and T.C.S. previously characterized and provided App knock-in mice; B.D.S. conceived and supervised the project, and wrote the manuscript. All the authors revised the manuscript and helped with comments and feedback.

Corresponding author

Correspondence to Bart De Strooper.

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Competing interests

B.D.S. is consultant for Jansen Pharmaceutics and receives research funding from them. The other authors do not have any competing financial interests to declare.

Extended data figures and tables

Extended Data Figure 1 Generation and characterization of Pld3ko mice.

a, Schema of the Pld3-targeted inactivation and lacZ-tagging strategy in Pld3ko mice. The trapping cassette containing a splicing acceptor (SA) followed by an IRES:lacZ was inserted between exons 9 and 10. Coloured bars indicate the primer-binding sites of quantitative PCR (qPCR) reactions (shown in b). IRES, internal ribosome entry site; neo, neomycin cassette; b, Validation of Pld3 inactivation by qPCR. The expression of exons 7 and 8 (e7-8) is strongly reduced, whereas exons 9 and 10 (e9-10) are not detectable (nd; expression value < 0.005) in Pld3ko mice that express lacZ instead at 1 month (e9-LacZ). Expression levels are relative to control. n = 6 for control and Pld3ko mice. ***P < 0.001, t-test. c, qPCR analysis of App mRNA and genes involved in its processing in Pld3ko mice relative to controls at 1 month. n = 6 for control and Pld3ko mice. d, X-gal staining of Pld3 heterozygous mice (Pld3het) brain shows the expression of Pld3 in neurons at 1 month. Fast red is used as a counterstain. cc, corpus callosum; Cx, cortex; DG, dentate gyrus; Hip, hippocampus; so, stratum oriens; sp, stratum pyramidalis; sr, stratum radiatum. Scale bar, 500 μm. Right panels (d’, d’’, d’’’) show magnified insets in stratum pyramidalis, stratum oriens and corpus callosum. Scale bars, 20 μm. d’, X-gal staining in CA1 pyramidal layer of the hippocampus. d’’, d’’’, β-galactosidase activity is not detectable in interneurons of the hippocampus (filled arrowheads in d’’) nor in the glia cells of the corpus callosum (open arrowheads in d’’’). e, Dorsal, lateral and central view of representative brains from control and Pld3ko mice at 3 months. h, height; l, length; t, thickness; w, width. f, Morphometric quantification of e. n = 2 control and n = 3 Pld3ko mice. Scale bars, 2 mm. g, Representative western blot of lysates from control and Pld3ko mice shows full-length APP, APP CTF and PLD3 expression in the hippocampus at 3 months. h, Densitometric analysis of western blot and ELISA quantification of Aβ40 and Aβ42 levels in the hippocampus from Pld3ko mice relative to controls. n > 7 control and n = 10 Pld3ko mice from three litters. i, ELISA of guanidine-HCl (GU)-soluble Aβ40 and Aβ42 levels. n = 8 control and Pld3ko; Appki mice. **P < 0.01, two-way ANOVA. Data are mean ± s.d. (b, c, f), and mean ± s.e.m. (h, i).

Extended Data Figure 2 Effect of wild–type and mutant PLD3 overexpression on APP.

a, ELISA shows Aβ40 and Aβ42 levels in the supernatant of HEK293T cells transfected to express GFP as a control, or wild-type PLD3, PLD3(K418R) (KR), or PLD3(V232M) (VM). Amyloid-β levels are expressed relative to control. n = 6 from 2 experiments. b, Representative western blots show full-length APP, APP CTF and PLD3 in lysates from HEK293T cells from the experiment in a. c, Densitometry analysis of western blots from the experiment in b. n = 6 from 2 experiments. d, Levels of Aβ40 and Aβ42 in the supernatant of HEK293T cells electroporated with 2.5, 0.75 and 0.25 μg of wild-type PLD3, PLD3(K418R) and PLD3(V232M) relative to GFP-electroporated control cells. n = 6 from 3 experiments. e, Representative western blots show full-length APP, APP CTF and PLD3 in lysates from d. For PLD3, short and long exposures of the same blot are shown. f, Densitometry of western blots from e, shown relative to control values. n = 6 from 3 experiments. All data are mean ± s.e.m. *P < 0.05, ***P < 0.001, two-way ANOVA.

Extended Data Figure 3 Pld3 regulates the morphology of lysosomes in vivo.

a, Confocal images show that PLD3 does not co-localize with EEA1 in early endosomes. The inset area is magnified in the middle and right panels. The product of the difference of the mean (PDM) image (right) illustrates the negative correlation of PLD3 and EEA1 (light blue arrowheads). Right bar shows colour codes for PDM values (negative in blue, positive in orange). Scale bars, 5 μm. b, Pearson’s coefficients for EEA1/PLD3 (n = 5) and LAMP1/PLD3 (n = 7). c, PLD3 is mostly localized in LAMP1-positive late endosomes/lysosomes. The inset area is magnified in the middle and right panels. The PDM image shows the positive correlation of PLD3 and LAMP1 staining (yellow arrowheads). Scale bars, 5 μm. Colour scales indicate PDM values. d, Mander’s coefficients for LAMP1 and PLD3 co-labelling. n = 7. e, Representative electron microscopy pictures of primary and secondary lysosomes (white and black arrowheads, respectively) from control and PLD3-deficient neurons. Arrow indicates electron-transparent inclusion in a secondary lysosome. Scale bars, 500 nm. f, Density, size and area occupied by primary lysosomes. For density and area: control, n = 50 fields from 2 brains; Pld3ko, n = 84 from 3 brains. For density: *P < 0.05, Mann–Whitney test. For area: ***P < 0.001, t-test. For size: control, n = 91; Pld3ko, n = 208; ***P < 0.001, t-test. g, Density, size and area occupied by secondary lysosomes. For density and area: control, n = 50 fields from 2 brains; Pld3ko, n = 84 from 3 brains. For density: *P < 0.05, Mann–Whitney test. For area: ***P < 0.001, t-test. For size: control: n = 22; Pld3ko, n = 73; *P < 0.05, t-test. All data are mean ± s.e.m.

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This file contains Supplementary Methods and the uncropped blots. (PDF 1958 kb)

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Fazzari, P., Horre, K., Arranz, A. et al. PLD3 gene and processing of APP. Nature 541, E1–E2 (2017).

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