| AP-2 is required for lysosomal targeting of BACE1.Figure 2(A, B) BACE1 protein levels in cortical lysates of WT and AP-2µ KO mice. Protein levels in the KO were normalized to the WT set to 100% (KO: 447.64±48.78%, p<0.000, N=7 biological replicates). Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. (C, D) Immunofluorescence levels of BACE1 are significantly increased in the cortex of AP-2µ KO mice when normalized to the WT set to 100% (KO: 162.714±11.082%, p=0.005, N=4, biological replicates). Scale bar: 100µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by one-sample Student's t‐test n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. (E) Localization of BACE1 in cultured WT and AP-2µ KO neurons. Scale bars: 20 µm, 5 µm (inserts). (F) Line plot of BACE1 immunofluorescence along the neurites Percentage of BACE1 puncta colocalizing with the LC3b (WT: 21.75±2.88, KO: 30.28±2.97, p=0.043, WT=41, KO=45 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within LC3b-positive organelles. Percentage of BACE1 puncta colocalizing with the LC3b (WT: 21.75±2.88, KO: 30.28±2.97, p=0.043, WT=41, KO=45 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within LC3b-positive organelles. 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. (I, J) Percentage of BACE1 puncta colocalizing with the eGFP-RAB7 (WT:19.54±1.91, KO: 37.09±3.13%, p<0.000, WT=44 and KO=43 neurons, N=4 biological replicates). Scale bar: 2µm. Arrowheads indicate BACE1 accumulation within RAB7-positive organelles. 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. (K, L) Colocalization of BACE1 and RAB7A in cortical sections of WT and AP-2μ ΚΟ brains, quantified using Pearson's colocalization coefficient (WT: 0.18±0.04, KO: 0.35±0.05, p=0.038, N=4 biological replicates). Scale bar: 10 µm. 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. (M Representative fluorescence images of WT neurons transfected with HA-BACE1-mCherry and immunostained for Cathepsin D. Channels were false color-coded to better illustrate the colocalization. Scale bars: 10µm. Arrowheads indicate BACE1 accumulation in lysosomes in the WT Dotted line outlines the neuron. N) Representative fluorescence images of AP-2µ KO neurons transfected with HA-BACE1-mCherry and immunostained for Cathepsin D. Channels were false color-coded to better illustrate the colocalization. Scale bars: 10µm. Arrowheads indicate BACE1 absence in the KO. Dotted line outlines the neuron. (O) Lysosomal localization of BACE1 is decreased in KO neurons (WT: 73.05±3.80, KO: 57.68±4.28, p=0.008, 45 WT and 44 KO 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. (P) Schematic illustration of BACE1 delivery to lysosomes monitored via BACE1-mKeima-Red. (Q, R) Representative fluorescence images of WT and AP-2μ KO neurons transfected with BACE1-mKeima-Red and co-transfected with eBFP to reveal the neuronal morphology. Scale bars: 10µm, 2µm in zoomed images. (S) The 550nm/438nm signal intensity ratio of BACE1-mKeima-Red quantified over the entire cell is significantly reduced in the KO condition compared to the WT (WT: 0.59±0.04, KO: 0.48±0.04, p=0.041, 28 WT and 25 KO neurons, N=3 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.