Sortilin and APP are allied <i>in vitro and in vivo</i>.

<p>(<b>A</b>) Colocalization of sortilin and APP. Mouse cortical neuron (upper) and brain cortex (lower) were immunostained for APP (red) and sortilin (green). Colocalization of sortilin and APP was indicated in merged panels (yellow). DAPI stained cell nuclei (blue). Plotted colocalization: 92% mean± SEM, n = 20, in cortical neurons and 95% mean± SEM, n = 3, in brain cortexes. Scale bar 25 µm for cortical neurons, 5 µm for enlarged image and 100 µm for brain cortex. (<b>B</b>) Co-IP of sortilin with APP in co-transfected HEK293 cells. HEK293 cells growing in 10 cm culture dishes were co-transfected with APP770-YFP/Sort-FL-myc/His (lane 1, 4) or Sort-T-myc/His (lane 2, 5). Cell lysates were immunoprecipitated with rabbit anti-GFP (α-GFP) for APP and blotted with mouse anti-Myc (α-Myc) for sortilin. Mixed lysates were used for IgG (lane 3). Sort-FL-myc.His (Sort-myc), Sort-T-myc.His (sort-T-myc) and APP770-YFP (APP-YFP) are indicated by arrows. (<b>C</b>) Co-IP of sortilin with APP in APPSwe/PS1dE9 transgenic mouse brain lysate. Mouse brain lysates were subjected to immunoprecipitation with rabbit anti-APP C’ (α-APP C’) and blotted with rabbit anti-sortilin (α-Sort) (left panel) or immunoprecipitation with α-Sort and blotted with α-APP C’ (right panel). Sortilin and APP are indicated by arrows. (<b>D</b>) Control for Co-IP using pEYFP. HEK293 cells were co-transfected with pEYFP/Sort-myc. Co-IP was performed using α-GFP and blotted with α-sort and α-GFP. Rabbit IgG (IgG) was used as a control for non-specific binding.</p

Effect of sortilin binding domain on APP lysosomal targeting.

<p>A: APP colocalization with lysosome. HEK293 cells were co-transfected with APP770-YFP and different sortilin constructs. The expressed APP and sortilin constructs excluding Sort-FL were visualized by either YFP or CFP fluorescence. Sort-FL-myc/His was immunostained with anti-sortilin, followed by staining with Cy3 conjugated secondary antibodies. Lysosomes were immunostained with lysosomal antibody (Lamp1), followed by staining with Cy5 conjugated secondary antibodies. B: Sortilin constructs sorting ASM to lysosomes. HEK293 cells were transfected with different sortilin constructs. The endogenous ASM was immunostained with mouse anti-ASM (Abcam), followed by staining with Cy3 conjugated secondary antibodies. Lysosome were immunostained with lysosomal antibody (Lamp1), followed by staining with Alexa 488 conjugated secondary antibodies. Sortilin constructs excluding Sort-FL were visualized by CFP fluorescence. Sort-FL-myc/His was immunostained with anti-sortilin, followed by staining with Cy5 conjugated secondary antibodies. Plotted colocalization is indicated at bottom. The percentage of colocalization is represented as mean± SEM (n = 20). The colocalization is compared with Sort-FL. The star (*) indicates <i>p</i><0.01. Scale bar 7.5 µm.</p

Lack of sortilin reduces APP distribution in lysosome and increases APP distribution in lipid rafts.

<p>Colocalization of APP with cell organelles in cortical neurons. Wild type (WT) and sortilin knockout (KO) mouse cortical neurons were immunostained for APP with mouse anti-APP-N’ (22c11) and followed by staining with Cy3 conjugated secondary antibodies (red) and cell organelles: Golgi, Early endosome, late endosome and lysosome immunostained with Giantin, EEA1, anti-mannose 6 phosphate receptor (for late endosome) and Lamp1, followed by staining with Alexa 488 conjugated secondary antibodies (green). Cell nuclei are stained by DAPI (blue). The colocalization is indicated in merged panels (yellow). Snapshots of colocalization are shown on the right of each panel. The percentage of colocalization (Col.) is plotted and is represented as mean± SEM (n = 20). The colocalization of APP with cell organelles is compared between WT and KO neurons. The star (*) indicates <i>p</i><0.01. Scale bar 10 µm.</p

Mapping APP binding to sortilin and the binding sequences.

<p>(<b>A</b>) Determining APP 1-287 and APP 713-770 binding to sortilin. HEK293 cells were co-transfected with sort-FL-myc and APP 1-287YFP or APP 1-542YFP or APP 541-671YFP or APP713-770YFP (lanes 1–4). Sort-T-myc was used for the co-transfection with the same APP-YFP constructs (lanes 5–8). Lysates from each co-transfection were used as input (lane 9–12). Cell lysates immunoprecipitated with α-Myc (lanes 1–8) and inputs were blotted with goat (gt)-α-GFP. The co-IP blot was re-probed with α-Myc for sortilin constructs (lower). APP 1-542YFP (87 kDa), APP 1-287YFP (58 kDa), APP 541-671YFP (42 kDa), APP 713-770YFP (33 kDa), Sort-myc (115 kDa) and Sort-T-myc (110 kDa) are indicated by arrows. (<b>B</b>) Determining N terminal binding sites between APP and sortilin. HEK293 cells were co-transfected with APP 1-287YFP and Sort 78-385CFP. Cell lysates were immunoprecipitated with α-APP-N’ for APP1-287YFP (lane 1) and mouse IgG (mIgG) as a control for non-specific binding (lane 2), and blotted with gt-α-GFP. Lysate was used as input (lane 3). APP1-287YFP (58 kDa) and Sort78-385CFP (61 kda) are indicated by arrows. (<b>C</b>) Determining APP 1-141 binding to sortilin. HEK293 cells were co-transfected with APP 1-141YFP and Sort-FL-myc. Cell lysates were immunoprecipitated with α-Myc for Sort-FL-myc (lane 1) and mIgG (lane 2), and blotted with gt-α-GFP for APP1-141YFP. The direct Immunoprecipitated APP1-141YFP by α-GFP was used as input (lane 3). Also, cell lysates were immunoprecipitated with rabbit-α-GFP for APP1-141YFP (lane 4) and rabbite IgG (rIgG) as control (lane 5), and blotted with α-Myc for sortilin. The direct immunoprecipitated Sort-FL-myc by α-Myc was used as input (lane 6). APP1-141YFP (41 kDa) and Sort-FL-myc (115 kDa) are indicated by arrows. (<b>D</b>) Determining C terminal binding sites between APP and sortilin. HEK293 cells were co-transfected with APP 713-770YFP and Sort del.MS2-CFP. Cell lysates were immunoprecipitated with α-APP-C’ for APP 713-770YFP (lane 1) and rIgG (lane 3), and blotted with α-Sort C’. Lysate was used as input (lane 2). Sort del.MS2-CFP (34 kDa) is indicated by arrow. (<b>E</b>) Determining Sort-MS1 (del.MS2) interaction with APP NPTYKFFE motif. Sort del.MS2-CFP was used with APP 713-770-YFP or APP 713-770 mut-YFP for the co-transfection of HEK293 and then subjected to FRET. APP 713-770 mut-YFP construct contains a mutated NPT<u>Y</u>KF<u>F</u>E motif where Y and F (underlined) are substituted with A. FRET efficiency representing the protein-protein interaction was determined from a photobleached region of interest (ROI). Three independent experiments were performed. Bars represent mean± SEM (n = 6 ROI×3). The star (*) indicates <i>p</i><0.01. Abbreviation: negative control: NC; positive control: PC.</p

Effect of sortilin on APP distribution in lipid rafts.

<p>A: HEK293 cells co-transfected with APP695 and sortilin constructs were lysed in Triton-X-100 buffer and subjected to discontinuous sucrose density gradient ultracentrifugation fractionation. Equal volumes from each fraction were examined by WB for APP and flotillin-1. B: Lack of sortilin increases APP distribution within lipid rafts in sortilin KO mice. Mouse brains were homogenized in Triton-X-100 buffer and subjected to discontinuous sucrose density gradient ultracentrifugation fractionation. Equal volumes from each fraction were examined by WB for APP and flotillin-1. Bars represent mean± SEM (n = 3) from three independent experiments. The star (*) indicates <i>p</i><0.01.</p

Lack of sortilin increases APP distribution in lipid rafts in cortical neurons.

<p>Wild type (WT) and sortilin knockout (KO) mouse cortical neurons were immunostained for APP with mouse anti-APP-N’ (22c11) and followed by staining with Cy3 conjugated secondary antibodies (red), and lipid rafts were immunostained with anti-flotillin, followed by staining with Alexa 488 conjugated secondary antibodies (green). Cell nuclei are stained by DAPI (blue). Colocalization is analyzed by counting the number of merged APP/raft lipids (yellow), and is plotted as mean of fold increase ± SEM (n = 20 neurons). The star (*) indicates <i>p</i><0.01. Scale bar 7.5 µm.</p

Effect of sortilin on APP lysosomal degradation.

<p>APP lysosomal targeting. HEK293 cells were co-transfected with APP-YFP and sortilin-pcDNA3.1 constructs or pcDNA3.1 (mock DNA) plasmid in 1∶1 molar ratio for 24 hours and then treated with DMSO or Bafilomycin AI, a lysosomal inhibitor, (BafA1, 4 µM, Sigma) for 6 h <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063049#pone.0063049-Kwon1" target="_blank">[30]</a>. Cell lysates were harvested and APP level was examined by WB with mouse anti-APP-N’ (22c11). Transfected APP-YFP served to monitor transfection efficiency. The APP level was plotted after correction as to the corresponding β-actin and transfection efficiency.</p