D495R BACE1 inhibited the cleavage of APP in Swedish APP overexpressing CHO cells and in SH-SY5Y cells.

<p>(A) Immunostaining analysis to examine the localizations of BACE1 and clathrin using an anti-myc antibody (2^nd lane) and an anti-clathrin antibody (3^rd lane). APPsw/Ncad-CHO cells were transfected either with WT BACE1 (middle row) or with D495R BACE1 (bottom row). Myc tag was linked to these BACE1 constructs. As a negative control, the cells were overexpressing empty vector (top row). The nucleus of the cell was labelled by DAPI (1^st lane). High-magnification images are shown in the 5^th lane. WT BACE1 was co-localized with clathrin in the cytoplasmic vesicles (arrowhead). On the other hand, D495R BACE1 was localized on the plasma membrane and hardly co-localized with clathrin. Scale bar indicates 10 μm. (B) APPsw/Ncad-CHO cells were transfected either with WT BACE1 (1^st and 3^rd lanes) or with D495R BACE1 (2^nd lane). Myc tag was linked to these BACE1 constructs. Equal amounts of cell lysates were immunoprecipitated by an anti-myc antibody, and then immunoblotted by an anti-clathrin antibody (upper panel, 1^st row), an anti-AP-2α antibody (2^nd row) and an anti-myc antibody (3^rd row). As a negative control, samples were immunoprecipitated by a normal rabbit IgG (3^rd lane). To confirm these results, samples were immunoprecipitated by an anti-clathrin antibody and immunoblotted by an anti-myc antibody (4^th row) or by an anti-clathrin antibody (5^th row). The expression levels of each protein in cell lysates are shown in the bottom panel. The level of BACE1/AP-2/clathrin complex was decreased in D495R BACE1 expressing cells compared with in WT BACE1 cells. (C) APPsw/Ncad-CHO cells were transfected either with WT BACE1 (1^st, 3^rd and 4^th lanes) or with D495R BACE1 (2^nd lane). These cells were lysed, and β-secretase activity in the cell lysates was measured by the β-secretase activity assay kit. All values were subtracted from the background readings obtained from only substrate (without secretase) (4^th lane). As a negative control, the CHO cells transfected WT BACE1 were treated with the BACE1 inhibitor which was included in the kit (3^rd lane). As a positive control, the substrate was mixed with the recombinant active BACE1which was also included in the kit (5^th lane). In vitro condition, the BACE1 activity in D495R BACE1 transfected cell was not different from that in WT BACE1 (n = 12, F _(4,31) = 32.48). n.s. indicates that there was no statistical significance. (D) APPsw/Ncad-CHO cells were transfected either with WT BACE1 (1^st lane) or with D495R BACE1 (2^nd lane). The levels of extracellular Swedish sAPPβ were determined by the Swedish sAPPβ specific ELISA kit. The levels of sAPPβ were normalized by the levels of BACE1 (mature plus immature forms) transiently overexpressed. The average level of sAPPβ in WT BACE1-transfected cells was regarded as 100% and that in D495R BACE1-transfected cells was relatively indicated. The level of Swedish sAPPβ in D495R BACE1-transfected cell was significantly smaller than that in WT BACE1 (n = 12, p = 0.034). * indicated p < 0.05. (E) Immunoblotting analysis using an anti-Aβ 6E10 antibody which only detects APP-CTFβ. APPsw/Ncad-CHO cells were transfected either with WT BACE1 (1^st lane) or with D495R BACE1 (2^nd lane). The maturation of WT BACE1 was not different with that of D495R BACE1. Although the band density of APP full length was not different, the band density of APP-CTFβ in D495R-transfected cells was significantly lower than that in WT BACE1 (n = 5, p = 0.009). * indicated p < 0.05. (F) APPsw/Ncad-CHO cells were transfected either with WT BACE1 (1^st lane) or with D495R BACE1 (2^nd lane). The levels of extracellular Aβ 40 were determined by the Aβ 40 specific ELISA kit. The average level of Aβ 40 in WT BACE1-transfected cells was regarded as 100% and that in D495R BACE1-transfected cells was relatively indicated. The level of Swedish Aβ 40 in D495R BACE1-transfected cell was significantly smaller than that in WT BACE1 (n = 12, p = 0.037). * indicated p < 0.05. (G) APPsw/Ncad-CHO cells were transfected either with WT BACE1 (1^st lane) or with D495R BACE1 (2^nd lane). The levels of extracellular Aβ 42 were determined by the Aβ 42 specific ELISA kit. The average level of Aβ 42 in WT BACE1-transfected cells was regarded as 100% and that in D495R BACE1-transfected cells was relatively indicated. The level of Swedish Aβ 42 in D495R BACE1-transfected cell was significantly smaller than that in WT BACE1 (n = 12, p = 0.003). * indicated p < 0.05. (H) Immunoblotting analysis using an anti-myc antibody. SH-SY5Y cells were transfected either with WT BACE1 (1^st lane) or with D495R BACE1 (2^nd lane). The maturation was analysed by the relative ration of the mature form of BACE1/the immature form of BACE1. The maturation of D495R BACE1 was significantly promoted compared to that of WT BACE1 (n = 6, p = 0.0001). * indicates p < 0.05. (I) SH-SY5Y cells were transfected either with WT BACE1 (1^st lane) or with D495R BACE1 (2^nd lane). The levels of extracellular WT sAPPβ were determined by the WT sAPPβ specific ELISA kit. The levels of sAPPβ were normalized by the levels of BACE1 (mature form) transiently overexpressed. The average level of sAPPβ in WT BACE1-transfected cells was regarded as 100% and that in D495R BACE1-transfected cells is relatively indicated. The level of WT sAPPβ in D495R BACE1-transfected cell was significantly smaller than that in WT BACE1 (n = 15, p = 0.0007). * indicates p < 0.05.</p

Overexpression of AP-2 promoted the cleavage of APP by BACE1 in Swedish APP overexpressing CHO cells and in SH-SY5Y cells.

<p>(A) APPsw/Ncad-CHO cells were transfected either with WT BACE1 and empty vector (1^st and 3^rd lanes) or with WT BACE1 and AP-2α (2^nd and 4^th lanes). Myc tag was linked to WT BACE1 and HA tag was linked to AP-2α. Equal amounts of cell lysate were immunoprecipitated by an anti-clathrin antibody, and then immunoblotted by an anti-myc antibody (upper panel, 1^st and 2^nd lanes). As a negative control, samples were immunoprecipitated by a normal mouse IgG (3^rd and 4^th lanes). The CHO cells overexpressing either with WT BACE1 and empty vector or with WT BACE1 and AP-2α were biotinylated, and cell surface level of BACE1 was examined by an anti-myc antibody (middle panel, 1^st row). As a positive control, cell surface level of TfR was examined (middle panel, 2^nd row). The expression levels of each protein in cell lysates are shown in the bottom panel. Overexpression of AP-2 increased the amount of BACE1/clathrin complex, and decreased the cell surface level of BACE1. (B) Immunostaining analysis to examine the localizations of BACE1 using an anti-myc antibody under AP-2 overexpressing conditions. APPsw/Ncad-CHO cells were transfected with WT BACE1 and AP-2α (right panel). As a negative control, the cells were overexpressing WT BACE1 and empty vector (left panel). Myc tag was linked to WT BACE1. WT BACE1 was localized on the cell surface (arrowhead) as well as in the intracellular compartments in a negative control cells. On the other hand, WT BACE1was hardly localized on the plasma membrane and mainly localized in the intracellular compartments under AP-2 overexpressing conditions. Scale bar indicates 10 μm. (C) Immunostaining analysis to examine the detailed localizations of BACE1 using anti-EEA1 and anti-Lamp2 antibodies under AP-2 overexpressing conditions. Myc tag was linked to WT BACE1. EEA1 was used as an endosome marker and Lamp2 was a lysosome marker. Under the AP-2 overexpressing condition, WT BACE1 was localized in the endosome and in the lysosome. (D) An internalization assay to examine the localization change of cell surface BACE1. APPsw/Ncad-CHO cells were transfected with WT BACE1 and empty vector (left panel) or with WT BACE1 and AP-2α (right panel). The cell surface BACE1 was labeled by an anti-myc antibody, followed by examination of its localization before (upper row) and 15 minutes after incubation (middle row). The expression of AP-2α was checked by an anti-HA antibody (bottom row). 15 minutes after incubation, the BACE1 labelled by an anti-myc antibody was observed on the cell membrane (arrowhead) and at the intracellular vesicles (arrow) in empty vector-transfected cells. On the other hand, the BACE1 was clearly accumulated in the intracellular compartment (arrow) and it was hardly observed on the cell membrane in the AP-2 overexpresing cells. (E) Triple immunostaining analysis for detecting BACE1/AP-2/clathrin complex in the internalization assay. APPsw/Ncad-CHO cells were transfected with WT BACE1 and AP-2α. Cell surface BACE1 was labelled by an anti-myc antibody. Clathrin and AP-2 were stained with anti-clathrin and anti-HA antibodies respectively. BACE1/AP-2/clathrin complex formed during the internalization of cell surface BACE1 (5 minutes after incubation) is indicated arrow. Right upper panel is a merged image and right bottom panel is high magnification of the image contained in the rectangle in the upper panel. Scale bar indicates 10 μm. (F) Immunostaining analysis to examine whether the internalized BACE1 co-localized with APP under AP-2 overexpressing conditions. Myc tag was linked to WT BACE1. The cell surface BACE1 was labelled by an anti-myc antibody, followed by incubating for 15 minutes. APP was stained by 6E10 antibody. 15 minutes after incubation, the BACE1 labelled by an anti-myc antibody was not clearly co-localized with APP. On the other hand, APP remarkably surrounded the compartment in which the BACE1 was accumulated. Scale bar indicates 10 μm. (G) APPsw/Ncad-CHO cells were transfected with WT BACE1 and empty vector (1^st lane), with WT BACE1 and AP-2α (2^nd lane) or with D495R BACE1 and AP-2α (3^rd lane). The levels of extracellular Swedish sAPPβ were determined by the Swedish sAPPβ specific ELISA kit. The levels of sAPPβ were normalized by the levels of BACE1 (mature form plus immature) transiently overexpressed. The average level of sAPPβ in WT BACE1 with empty vector-transfected cells was regarded as 100% and that in other cells was relatively indicated. The level of Swedish sAPPβ in WT BACE1 and AP-2α-transfected cell was significantly higher than that in WT BACE1 and empty vector (n = 12, F _(2,33) = 7.86, p = 0.001). On the other hand, the level in D495R BACE1 and AP-2α-transfected cell was lower than that in WT BACE1 and AP-2α-transfected cell (p = 0.002). * indicates p < 0.05. (H) SH-SY5Y cells were transfected with WT BACE1 and empty vector (1^st lane), with WT BACE1 and AP-2α (2^nd lane) or with D495R BACE1 and AP-2α (3^rd lane). The levels of extracellular WT sAPPβ were determined by the WT sAPPβ specific ELISA kit. The levels of sAPPβ were normalized by the levels of BACE1 (mature form) transiently overexpressed. The average level of sAPPβ in WT BACE1 with empty vector-transfected cells was regarded as 100% and that in other cells was relatively indicated. The level of WT sAPPβ in WT BACE1 and AP-2α-transfected cell was significantly higher than that in WT BACE1 and empty vector (n = 14, F _(2,39) = 6.34, p = 0.045). On the other hand, the level in D495R BACE1 and AP-2α-transfected cell was lower than that in WT BACE1 and AP-2α-transfected cell (p = 0.001). * indicates p < 0.05.</p

HFD promoted BACE1/clathrin complex formation in APP transgenic mice.

<p><i>(A)</i> Equal amounts of brain samples from 7–8 months age of the control APP mice and APP-HFD mice (fed with HFD for 20 weeks) were immunoprecipitated by an anti-BACE1 antibody, and then SDS-PAGE was conducted. As a negative control, samples were immunoprecipitated by a normal mouse IgG. To visualize proteins, the gels were stained with silver nitrate. These proteomics assays were repeated twice using different four brain samples from the control APP mice as well as four samples from APP-HFD mice. The band density around 180 kDa was significantly higher in APP-HFD mice than that in the control APP mice. The protein bands were excised and subjected to in gel trypsinization, and molecular mass analysis of the tryptic peptides indicated that the band was clathrin heavy chain. (<i>B)</i> Equal amounts of brain samples from 7–8 months age of the control APP mice and APP-HFD mice (fed with HFD for 20 weeks) were immunoprecipitated by a monoclonal anti-BACE1 antibody (2^nd row) and then they were immunoblotted by an anti-clathrin antibody or by an anti-BACE1 antibody. As a negative control, samples were immunoprecipitated by a normal mouse IgG (top row). These immunoprecipitation assays were repeated twice using different four brain samples from the control APP mice as well as four samples from APP-HFD mice. HFD promoted the BACE1/clathrin complex formation in APP transgenic mice. <i>(C</i>) Immunoblotting analysis using an anti-clathrin antibody. Statistical analysis is shown in the bottom panel. The band density of the clathrin heavy chain was normalized by that of β-actin. The band density of the control APP mice was regarded as 100% and that of APP-HFD mice was relatively indicated. The band density of clathrin in APP-HFD mice (fed with HFD for 20 weeks) was the same as that in the control APP mice. n.s. indicates that there was no statistical significance.</p