| AP-2 regulates BACE1 recycling and is mostly dispensable for BACE1 endocytosis.Figure 1(A) BACE1 internalization kinetics after 20 minutes (WT: 19.79±1.89%, KO: 13.28±1.57%, p WT vs KO=0.017) and 40 minutes of HA antibody chase (WT: 15.99±2.04, KO: 9.29±1.33, pWT vs KO=0.010) is significantly decreased in AP-2µ KO neurons (27 WT and 29 KO neurons). Data information: All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. (B) Representative examples of internalized BACE1 after 5 min of HA antibody pulse in HA-BACE1-eGFP-overexpressing WT and KO neurons. Scale bar: 5µm. (C, D) Internalized BACE1 levels after 20 min of HA antibody pulse in HA-BACE1-eGFP-overexpressing WT and KO neurons, calculated as the HA (internalized)/eGFP (total) signal intensity ratio (WT: 0.50±0.052, KO: 0.23±0.036, p=0.000, 30 WT and 36 KO neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square. All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. (E, F) Recycled BACE1 levels after 20 min of HA antibody pulse in HA-BACE1-eGFP-overexpressing WT and KO neurons, calculated as the HA(recycled)/eGFP(total) signal intensity ratio (WT: 0.29±0.031, KO: 0.53±0.040, p<000, 30 WT and 32 KO neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square. All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. Recycled levels of BACE1 after 20 min of HA antibody pulse in HA-BACE1-mCherry-transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N pWTRAB4 vs pWTRAB4S22N>0.999, 34-38 neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels , where analyzed BACE1 fraction is marked with the red square. Recycled levels of BACE1 after 20 min of HA antibody pulse in HA-BACE1-mCherry-transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N, calculated as the HA(recycled)/mCherry(total) signal intensity ratio (WTRAB4: 0.14±0.02, KORAB4: 0.20±0.02, WTRAB4-S22N: 0.14±0.02, KORAB4S22N:0.14±0.01, pWTRAB4 vs pKORAB4=0.033, pKORAB4 vs pKORAB4S22N=0.035, pWTRABS22N vs pKORABS22N=0.999, pWTRAB4 vs pWTRAB4S22N>0.999, 34-38 neurons, N=4 biological replicates). Scale bar: 5 µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. Recycled levels of BACE1 after 20 min of HA antibody chase in HA-BACE1-mCherry-transfected WT and KO neurons, co-expressing either eGFP-RAB11 or eGFP-RAB11-S22N, calculated as the HA(recycled)/mCherry(total) signal intensity ratio (WTRAB11: 0.11±0.01, KORAB11: 0.29±0.02, WTRAB11A-S25N: 0.14±0.02, KORAB11A-S25N: 0.19±0.02, pWTRAB11 vs pKORAB11<0.0001, pWTRAB11 vs pWTRAB11A-S25N=0.761, pKORAB11 vs pKORAB11A-S25N=0.001, pWTRAB11A-S25N vs pKORAB11A-S25N=0.175, 28-37 neurons per condition, N=3 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA-BACE1-mCherry transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N pWTRAB4 vs pWTRAB4S22N=0.450, 27-37 neurons per condition, N=3 biological replicates). Scale bar: 5 µm. Data information: Experimental designs are depicted in the top panels where analyzed BACE1 fraction is marked with the red square. Internalized levels of BACE1 after 20 minutes of HA antibody uptake in HA-BACE1-mCherry transfected WT and KO neurons, co-expressing either eGFP-RAB4 or eGFP-RAB4-S22N, calculated as the HA(internalized)/mCherry(total) signal intensity ratio (WTRAB4: 0.54±0.05, KORAB4: 0.33±0.02, WTRAB4-S22N: 0.63±0.06, KORAB11A-S25N: 0.50±0.04, pWTRAB4 vs pKORAB4=0.004, pKORAB4 vs pKORAB4S22N=0.046, pWTRABS22N vs pKORABS22N=0.182, pWTRAB4 vs pWTRAB4S22N=0.450, 27-37 neurons per condition, N=3 biological replicates). Scale bar: 5 µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by ANOVA n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. (L, M) BACE1 surface levels are increased in AP-2µ KO neurons, when compared to the WT set to 100% (KO: 129.65±5.30%, p=0.005, N=4 biological replicates). (N) Surface versus total BACE1 ratio is not altered in AP-2µ KO neurons when normalized to the WT set to 100% (KO: 83.02±10.93%, p=0.109, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test in (M,N). n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. (O, P) Accumulation of endogenous BACE1 in RAB5-positive early endosomes, quantified using Pearson's colocalization coefficient (WT: 0.41±0.01, KO: 0.46±0.02, pWT vs pKO=0.021, 58 WT and 54 KO neurons, N=4 biological replicates). Scale bar: 10 µm. Arrows indicate BACE1 accumulation within RAB5-positive endosomes. Data information All graphs show mean ± SEM, statistical analysis was performed by unpaired two-tailed Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. (Q) Schematic illustration depicting the role of AP-2 in BACE1 endosomal trafficking.