Adaptor protein sorting nexin 17 regulates amyloid precursor protein trafficking and processing in the early endosomes

Abstract

Accumulation of extracellular amyloid beta peptide (Abeta), generated from amyloid precursor protein (APP) processing by beta- and gamma-secretases, is toxic to neurons and is central to the pathogenesis of Alzheimer disease. Production of Abeta from APP is greatly affected by the subcellular localization and trafficking of APP. Here we have identified a novel intracellular adaptor protein, sorting nexin 17 (SNX17), that binds specifically to the APP cytoplasmic domain via the YXNPXY motif that has been shown previously to bind several cell surface adaptors, including Fe65 and X11. Overexpression of a dominant-negative mutant of SNX17 and RNA interference knockdown of endogenous SNX17 expression both reduced steady-state levels of APP with a concomitant increase in Abeta production. RNA interference knockdown of SNX17 also decreased APP half-life, which led to the decreased steady-state levels of APP. Immunofluorescence staining confirmed a colocalization of SNX17 and APP in the early endosomes. We also showed that a cell surface adaptor protein, Dab2, binds to the same YXNPXY motif and regulates APP endocytosis at the cell surface. Our results thus provide strong evidence that both cell surface and intracellular adaptor proteins regulate APP endocytic trafficking and processing to Abeta. The identification of SNX17 as a novel APP intracellular adaptor protein highly expressed in neurons should facilitate the understanding of the relationship between APP intracellular trafficking and processing to Abeta.

FIGURE 1.

Sorting nexin 17 binds to the APP cytoplasmic domain through the YXNPXY motif. A, a schematic diagram of the APP cytoplasmic domain and GST pulldown assay. GST or GST-APP fusion proteins were incubated with lysates from U87 cells stably expressing full-length SNX17 and subjected to Western blotting with anti-Myc antibody. GST fused with the wild-type (WT) APP cytoplasmic domain but not YXNPXY mutants interact with SNX17. B, a schematic diagram depicting full-length and PX domain-deleted (ΔPX-SNX17) SNX17 and GST pulldown assay. SNX17 contains a PX domain at the N terminus, a FERM (protein 4.1, ezrin, radixin, and moesin)-like domain, and a C-terminal region with undefined function. The PX domain of SNX17 is not required for SNX17 binding to the APP cytoplasmic tail. GST or GST-APP was incubated with lysates from U87 cells stably expressing full-length SNX17 or ΔPX-SNX17 and subjected to Western blotting with anti-Myc antibody. GST-APP, but not GST, binds to both full-length SNX17 and ΔPX-SNX17. TM, transmembrane domain.

FIGURE 2.

The dominant-negative mutant of SNX17 reduces steady-state levels of APP and increases Aβ production. A, ΔPX-SNX17 decreases cellular APP levels. HA-APP was cotransfected with full-length SNX17 or ΔPX-SNX17, and cellular APP levels were examined by Western blotting using anti-HA antibody. Expressions of SNX17 and ΔPX-SNX17 were confirmed by Western blotting using anti-Myc antibody. B, ΔPX-SNX17 increases Aβ production. HA-APP was cotransfected with full-length SNX17 or ΔPX-SNX17, and Aβ40 and Aβ42 levels in the conditioned medium were analyzed by ELISA. The Aβ levels were normalized by expression of green fluorescent protein, cotransfected with APP and SNX17 (not shown). ^**, p < 0.01 compared with the controls.

FIGURE 3.

SNX17 regulates APP cell surface levels, turnover, and Aβ production. A, SNX17 knockdown decreases cellular APP levels. Control or SNX17 siRNA oligonucleotides were cotransfected with HA-APP into U87 cells, and the levels of APP were examined by Western blotting with anti-HA antibody. Quantification of SNX17, APP, and actin Western blots is shown in the right panel. B, the cellular level of APP was restored when an siRNA oligonucleotide-resistant SNX17 construct was cotransfected with SNX17 siRNA. Western blot analysis was performed using U87 cells cotransfected with HA-APP, SNX17 siRNA, and either control vector or siRNA-resistant SNX17 construct. C, SNX17 knockdown decreases APP half-life. U87 cells were cotransfected with HA-APP and either control or SNX17 siRNA. After 48 h, cells were incubated with cycloheximide for 0, 15, 30, 45, or 60 min, followed by Western blotting with anti-HA antibody. D, SNX17 knockdown decreases APP half-life, detected by pulse-chase analysis. U87 cells were cotransfected with HA-APP and either control or SNX17 siRNA. After 48 h, cells were incubated with medium containing [³⁵S]Met/Cys for 30 min and chased with medium without [³⁵S]Met/Cys for 0, 30, 60, or 120 min, followed by immunoprecipitation with anti-HA antibody. Radiolabeled APP bands were visualized after SDS-PAGE. E, SNX17 knockdown increases Aβ production. Aβ40 and Aβ42 levels in the conditioned medium from U87 cells cotransfected with HA-APP and either control or SNX17 siRNA were analyzed by ELISA. ^*, p < 0.05 compared with the controls.

FIGURE 4.

APP and SNX17 colocalize in the early endosomes. U87 cells transiently transfected with HA-APP were incubated with anti-HA IgG at 37 °C for 1 h, fixed, and then stained with anti-SNX17 (A and B) or anti-EEA1 (D and E), followed by secondary antibodies. APP is found in both SNX17- and EEA1-positive compartments (C and F). SNX17 and EEA1 are highly colocalized (G-I). Arrows highlight staining with significant colocalization.

FIGURE 5.

SNX17 is highly expressed in mouse brain and colocalizes with APP in primary neurons. A, SNX17 is highly expressed in several regions of adult mouse brain where APP and LRP1 are also highly expressed. B, SNX17 is highly expressed in primary cultured cortical and hippocampal neurons. C, SNX17 and APP are colocalized in primary hippocampal neurons. Primary hippocampal neurons were co-stained with anti-SNX17 and anti-APP antibodies. A region including several neuronal processes is shown in higher magnifications. Arrows highlight staining with significant colocalization.

FIGURE 6.

Dab2 mediates APP endocytosis. A, Dab2 directly interacts with APP through the NPXY motif. GST, GST-Dab2, or GST-Dab2 PTBPTB (tandem PTB) was incubated with lysates from CHO LRP-null cells transiently transfected with wild-type HA-APP or mutants and subjected to Western blotting with anti-HA antibody. GST-Dab2 protein binds to wild-type APP but not to NPXY mutants. Stronger binding to APP was detected with the tandem PTB domain construct compared with full-length Dab2. B, Dab2 facilitates APP endocytosis. Endocytosis of APP, measured 15 min after 37 °C incubation, is increased in Dab2-transfected cells and decreased in the dominant-negative mutant-containing PTB domain-transfected cells. C, the cell surface levels of APP are increased in cells transfected with the PTB domain. CHO LRP-null cells stably transfected with control vector or the Dab2 PTBPTB domain were analyzed for the ratios of cell surface and total APP. D, full-length Dab2 increases, whereas the Dab2 PTB domain decreases, Aβ production. Aβ40 and Aβ42 levels in the conditioned medium from U87 cells cotransfected with HA-APP and pcDNA3, full-length Dab2, or the Dab2 PTB domain were analyzed by ELISA. ^**, p < 0.01 compared with the controls.