SNX6 expression in mouse brain. A) Immunohistochemical analyses in brain tissue of C57Bl/6 mice show neuronal distribution in the cortical layers. B–G) Regional distribution of SNX6 in mouse brain: front parietal cortex (B), striatal cortex (C), entorhinal cortex (D), subiculum (E), medial septal nucleus (F), and hippocampus (G; CA). H–J) Subcellular colocalization with Rab5: SNX6 (H), Rab5 (I), and overlay (J). Note the predominant neuronal expression of SNX6 in indicated regions of the mouse brain.

SNX6 negatively regulates the retrograde transport of BACE1. A) BACE1 antibody uptake assay was performed using the cell lines used in Fig. 2A. Cells were transfected with BACE1. After 1 d of incubation, BACE1 at the cell surface was labeled with anti-BACE1 (N-terminal) antibody and subsequently chased for indicated time periods. Representative confocal images are shown. Scale bar = 10 μm. B) Quantitative analysis of BACE1 subcellular localization; 125 cells/case, on average, were examined for degree of perinuclear accumulation near the TGN. Categories used for scoring degree of accumulation: little, partial, prominent, and exclusive. C) Percentages of cells that showed dominant perinuclear accumulation (=prominent+exclusive) were plotted at each chase time (n=3). Values are means ± sd. *P < 0.05, **P < 0.001; 2-tailed t test.

Identification of SNX6 as a BACE1-interacting protein. A) Coomassie staining of the components of BACE1-harboring protein complexes from a control line and a cell line stably expressing BACE1-TAP. Bands were excised from the gel and analyzed by mass spectrometry. Band and peptide sequences corresponding to SNX6 are shown. B) Schematic representation of SNX6 protein containing PX and BAR (CC) domains and 2 deletion constructs (ΔPX and ΔBAR). Amino acid numbers are shown. C) Coimmunoprecipitation (CO-IP) of SNX6 with BACE1. Cross-linked lysates prepared from HeLa cells expressing mock (lane 1), HA-tagged SNX6 (lane 2; FL), ΔPX (lane 3), and ΔBAR (lane 4) were subjected to the immunoprecipitation using anti-myc antibodies to pull down BACE1. After reversal of cross-linking, ∼60-kDa HA-SNX6 was detected by anti-HA antibody. IgG-HC and IgG-LC denote immunoglobulin heavy- and light-chain bands, respectively.

Proteomic identification of SNX6 as a component of BACE1-harboring protein complex. A) Schematic representation of BACE1-TAP protein and purification strategy. TAP tag consists of the calmodulin binding domain, TEV protease cleavage site, and protein A, and was fused to the C terminus of BACE1. Inactive BACE1-TAP was generated by introducing a missense mutation (D92G) at the active site. B) Western blot analysis of SY5Y cells stably expressing either control vector (C) or BACE1-TAP (WT). Elevated C99 levels were observed in the stable SY5Y cells expressing BACE1-TAP. C99 was detected by 6E10 antibody, and asterisk denotes nonspecific bands. C) TAP process of BACE1-harboring complex prepared from formaldehyde (FA)-cross-linked SY5Y cell lysates. Samples from each purification step were analyzed by Western blotting using anti-BACE1 antibody. BACE1-TAP and BACE1 (D92G)-TAP cross-linked to higher molecular complexes.

Reducing SNX6 increases basal BACE1 levels. A) Reducing SNX6 increases endogenous BACE1 levels. Neuro2a cells stably expressing the Swedish variant of APP were transfected with either nonsilencing shRNA vector (NS) or shRNA against mouse SNX6 (SNX6). After 2 d incubation, cell lysates were subjected to Western blot analysis. B) Quantification of BACE1 was performed using NIH ImageJ (n=3). Values are means ± sd. *P < 0.005; 2-tailed t test. C) Reducing SNX6 does not affect BACE1 mRNA levels. HeLa cells were transfected with 2 distinct SNX6 shRNAs (SNX6 RNAi) or negative control shRNAs (NS). Real-time PCR was performed, and mRNA levels of SNX6 and BACE1, normalized to β-actin mRNA, were quantified. D) Pulse-chase experiments using Halo-tagged BACE1. HEK293 cell lines stably expressing either nonsilencing shRNA vector (NS) or shRNA against human SNX6 (SNX6; see Fig. 4A) were transfected with Halo-tagged BACE1. After 1 d of incubation, cells were labeled with biotin-conjugated Halo-tag ligand and chased for indicated times. BACE1 turnover was analyzed with Western blot analysis. Representative blots are shown. E) Protein levels of BACE1 were plotted with the chased times. Error bars = sd from 3 experiments.

Reducing SNX6 increases BACE1-mediated cleavage of APP. A) SNX6 expression in stable HEK293 clonal cell lines transfected with either nonsilencing shRNA vector (NS) or shRNA against human SNX6 (SNX6). B) Immunoprecipitation and Western blot analysis of sAPPβ and sAPPα, secreted from the HEK293 stable cells in A that were transfected with APP. At 1 d post-transfection, conditioned medium collected after 3 h of incubation was subjected to immunoprecipitation using either BACE1 cleavage site-specific antibody (sβwt) or 6E10, followed by Western blot analysis with LN27 to detect sAPPβ and sAPPα, respectively. C, D) Quantification of the sAPPβ (C) and sAPPα (D) bands (normalized to full-length APP) was performed using infrared-based quantitation (LI-COR) and combining >6 independent experiments. E) Detection of a major BACE1-derived C-terminal fragment (C99) and α-secretase-derived C83. HEK293 stable cells in A were transfected with APP. After 1 d of incubation with compound E to inhibit γ-secretase activity, cell lysates were subjected to Western blot analyses using indicated antibodies. F, G) Quantification of the C99 (F) and C83 (G) bands (normalized to full-length APP) was performed using infrared-based Western blot analysis (LI-COR) and combining 4 independent experiments. H) Reducing SNX6 increases the secretion of Aβ_1–40. Conditioned medium was subjected to sandwich ELISA to quantify Aβ_1–40 (n=3). Values are means ± sd. *P < 0.0005, **P < 0.00005, ***P < 0.005; 2-tailed t test.