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, 18 (1), 26-37

Overexpression of SNX3 Decreases Amyloid-β Peptide Production by Reducing Internalization of Amyloid Precursor Protein

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Overexpression of SNX3 Decreases Amyloid-β Peptide Production by Reducing Internalization of Amyloid Precursor Protein

Shaohua Xu et al. Neurodegener Dis.

Abstract

Background: Sorting nexins (SNXs) have diverse functions in protein sorting and membrane trafficking. Recently, single-nucleotide polymorphisms in SNX3 were found to be associated with Alzheimer disease. However, it remains unknown whether SNX3 participates in amyloid (A)β peptide production.

Objective: To examine the role of SNX3 in Aβ production and APP processing.

Methods: The effect of increased expression of SNX3 was studied in HEK293T cells. Aβ peptides were measured by immunoassay. Protein-protein association was analyzed by a bimolecular fluorescence complementation (BiFC) assay. APP uptake was measured with an α-bungarotoxin-binding assay, and flow cytometry was used to measure cell surface APP levels.

Results: We found that overexpression of SNX3 in HEK293T cells decreases the levels of secreted Aβ and soluble N-terminal APP fragments (sAPPβ). The reduction correlated with a decreased association of APP with BACE1, as revealed by BiFC. This effect may, in part, be explained by a reduced internalization of APP; SNX3 overexpression reduced APP internalization as determined by an α-bungarotoxin-binding assay, and caused increased APP levels on the cell surface, as shown by flow cytometry. In addition, SNX3 overexpression increased the cellular levels of full-length APP.

Conclusion: These results provide evidence that SNX3 regulates Aβ production by influencing the internalization of APP.

Keywords: Alzheimer disease; Amyloid peptide; Amyloid precursor protein; Secretase; Sorting nexin.

Conflict of interest statement

Disclosure Statement

The authors declare that there are no conflicts of interest.

Figures

Fig. 1
Fig. 1
Overexpression of SNX3 reduces the production of Aβ peptides. For the Western blot and MSD measurement, HEK293T cells were cotransfected with empty vector p3×FLAG-CMV-10, SNX3, or SNX29 together with Myc-APP. a The expression of endogenous and overexpressed SNX3 was confirmed by Western blot with anti-SNX3 (sc-10619). The blot shown is representative of 4 independent experiments. b The distribution of endogenous and overexpressed SNX3 was examined by immunocytochemistry with anti-SNX3 (sc-376667). Representative images from 2 independent experiments are shown. To avoid saturation, the confocal image of SNX3 labeling in the cells overexpressing SNX3 was scanned with a lower detection gain than that in the empty vector-transfected cells. Scale bar, 10 μm. Levels of secreted Aβ40 (c), Aβ42 (d), and sAPPβ (e) were measured in the conditioned medium of cells overexpressing SNX3 or SNX29, and normalized to the levels in the “empty vector” control group. f SH-SY5Y_APP cells were transfected with empty vector or SNX3. Levels of SNX3 in cell lysates were detected by Western blot with anti-SNX3 (sc-10619). g Quantification of SNX3. h Levels of secreted Aβ40 in the conditioned medium. Mature and immature BACE1 levels were examined by Western blot (I), and quantifications of mature (j) and immature BACE1 (k) are shown. SNX3 was detected with anti-SNX3 (sc-10619) antibody. One-sample t test was performed from at least 4 independent experiments for all the statistical analyses. ns, not significant. *p < 0.05, **p < 0.01.
Fig. 2
Fig. 2
Overexpression of SNX3 decreases the association between APP and BACE1 in intact cells. a Schematic diagram of BiFC constructs. b, c Western blot was performed to confirm the expression and cleavage of APP constructs. Lane 1: nontransfected HEK293T cells. Lane 2–5: HEK293T cells transfected with different constructs as indicated in the figures. Full-length APP was detected with antibody 22C11 (b), and APP CTFs were detected with antibody Y188 (c). Representative blots from 2 independent experiments are shown. d Representative images of cells labeled with nuclei staining (blue), expressing mCherry (red) and emitting BiFC signals (green) in each experimental condition. Scale bar, 100 μm. e Quantification of BiFC signals from 4 independent experiments. The APP/BACE1 association in “empty vector” control cells was normalized to 1. One-sample t test was performed. *p <0.05, ***p < 0.001.
Fig. 3
Fig. 3
Overexpression of SNX3 inhibits APP internalization and increases APP levels on the cell surface. a Characterization of APP internalization. HEK293T cells were transfected with BBS-APP, and cell surface BBS-APP was labeled by incubation with Alexa Fluor 555-conjugated α-bungarotoxin at 4 ° C for 20 min. Then cells were incubated at 37 ° C for different time periods (0, 10, and 30 min). Representative images of APP labeling (red) and nuclei staining (blue) from each time point are shown. Scale bar, 10 μm. b, c HEK293T cells were cotransfected with empty vector (pEGFP-C1) or pEGFP-C1_SNX3 and BBS-APP, and internalization of cell surface APP was examined. Scale bar, 10 μm. d APP fluorescent vesicular structures with the same size range and circularity were quantified. More than 20 cells from 3 independent experiments were imaged and analyzed for every time point of each condition. One-way ANOVA followed by Bonferroni’s post hoc test was used for the statistical analysis. e–g Cell surface levels of APP were determined by flow cytometry. e Representative overlay plot of cell surface labeling of APP. f The geometric mean fluorescence intensity (MFI) of cell surface APP was quantified. g Quantification of the percentages of surface APP-labeled cells. Four independent experiments were performed. Two-tailed unpaired t test was performed. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig. 4
Fig. 4
Overexpression of SNX3 increases the level of full-length APP. a–e HEK293T cells were cotransfected with Myc-APP and either empty vector or FLAG-SNX3. Western blot was performed to examine the level of full-length APP in cell lysates, with GAPDH used as a loading control. a, b APP was detected with anti-Myc antibody. c–e Mature and immature APP was revealed by anti-APP (22C11) antibody. Anti-SNX3 (sc-10619) antibody was used to detect SNX3. Quantitative analysis of the APP levels normalized to that in control cells expressing empty vector. At least 3 independent experiments were performed. One-sample t test was performed. *p < 0.05.

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