| AP-2 regulates axonal transport of BACE1.Figure 3(A, B) Representative fluorescence images (A) and corresponding kymographs (B) from time-lapse videos of control neurons transfected with HA-BACE1-eGFP and co-transfected with AP-2μ-mCherry. Scale bars: (A) 2μm, 1.6µm inserts, (B) x=2μm, y=5s. Arrowheads in (A) correspond to BACE1 carriers indicated by the same arrowheads in (B). (C) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT and AP-2μ KO neurons transfected with HA-BACE1-eGFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels. (D) Loss of AP-2μ significantly decreases the BACE1 motility (WT: 37.42±3.79%, KO: 23.07±3.07%, p=0.003, WT=31, KO=32 neurons, N=4 biological replicates). 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. (E) Loss of AP-2μ significantly decreases the anterograde and retrograde BACE1 velocity (WTAntero: 0.37±0.02 μm/s, KOAntero: 0.22±0.03 μm/s, pWTAntero vs pKOAntero<0.000; WTRetro: 0.35±0.02 μm/s, KORetro: 0.25±.03 μm/s, pWTRetro vs pKORetro =0.006, WT=35 and KO=32 neurons, N=4 biological replicates). 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. (F) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT and AP-2μ KO neurons co-transfected with HA-BACE1-GFP and AP-2μ-mRFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels. (G) Bi-directional mobility of BACE1 carriers in WT and AP-2μ ΚΟ neurons, co-expressing the AP-2μ-mRFP (WT: 37.60±2.20%, KO: 25.61±2.67%, WTAP-2μ-mRFP: 45.04±3.27%, KOAP-2μ-mRFP: 40.07±2.37%, pWT vs KO=0.005, pKO vs KO+AP-2μ-mRFP<0.000, pWT vs WT+AP-2μ-mRFP=0.186, pWT+AP-2μ-mRFP vs KO+AP-2μ-mRFP=0.570, 43-53 neurons per condition, N=4 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in (G). n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. (H) Representative kymographs of WT and AP-2μ KO neurons co-expressing the HA-BACE1-GFP and tagRFP-LC3B. Scale bars: x=2μm, y=5s. (I) Reduced bi-directional mobility of BACE1-containing autophagosomes in AP-2µ KO neurons. (WT: 33.53±4.19%, KO: 19.45±5.14%, p=0.000, WT=37 and KO=35 neurons, N=4 biological replicates). 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. (J) Representative kymographs of WT neurons co-expressing HA-BACE1-GFP and AP2-αΑ WT or AP2-αΑ Μut. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels. (K) Mobility of BACE1 carriers is significantly decreased in neurons overexpressing the AP2-αΑ Mut compared to neurons expressing the AP2-αΑ WT (WT: 52.23±4.16%, Mut: 27.95±3.31, p=<0.000, WT=26 and Mut=29 neurons, N=4 biological replicates). 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. (L) Representative fluorescence images and corresponding kymographs from time-lapse videos of WT neurons transfected with either HA-BACE1-GFP or HA-BACE1-LL/AA-GFP. Scale bar: 5µm top panels, x=2μm, y=5s bottom panels. (M) Reduced mobility of BACE1-LL/AA-positive vesicles, when compared to WT BACE1 (WT: 38.97±1.40%, LL/AA: 19.89±1.77%, p<0.000, WT=31 and LL/AA=33 neurons, N=4 biological replicates). 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. (N) Reduced velocity of BACE1-LL/AA-positive vesicles, when compared to WT BACE1 (WTAntero: 0.38±0.03 μm/s, LL/AAAntero: 0.15±0.03 μm/s, pWTAntero vs pLL/AAAntero<0.000; WTRetro: 0.39±0.03 μm/s, LL/AARetro: 0.25±.03 μm/s, pWTRetro vs pLL/AARetro =0.000, WT=31 and LL/AA=33 neurons, N=4 biological replicates). 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. (O) Schematic illustration of how AP-2 regulates BACE1 intracellular trafficking and transport in neurons.