| AP-2 prevents amyloidogenic processing of APP in neurons.Figure 4(A, B) The eGFP/mCherry signal intensity ratio is significantly increased in mCherry-APP-P1-eGFP-expressing KO neurons comparing to the WT (WT: 0.94±0.06%, KO: 1.65±0.18, p=0.000, WT=39 and KO=41 neurons, N=5 biological replicates). Scale bars: 2μm. 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. Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 100 µm (C) Confocal images of WT and AP-2μ ΚΟ entorhinal cortex immunostained with antibody recognizing total APP and APP-CTFs. Scale bars: 20µm (D). (E-G) Levels of APP-CTFs are significantly increased in AP-2µ KO cortex compared to the WT set to 100% (KOC99: 156.65±8.37%, p=0.004, N=4 biological replicates; KOC83: 154.79±15.6%, p=0.012; N=5 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. (H) Levels of full-length (FL) APP are unaltered in AP-2µ KO cortex (KOAPP FL: 106.64±12.96%, p=0.315, N=6 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. (I) Levels of Nicastrin (NCT) are unaltered in the AP-2µ KO cortex (J, K) Intracellular levels of Aβ1-40 are significantly increased in AP-2µ KO axons compared to the WT (WTAβ1-40: 1.82±0.12, KOAβ1-40: 2.45±0.12, p=0.000, WT=24 and KO=24 neurons, N=3 biological replicates). Scale bars, 5µm. 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. Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 µm. Intracellular levels of Aβ1-42 are significantly increased in AP-2µ KO axons (WTAβ1-42: 0.65±0.08, KOAβ1-42: 0.91±0.10, p=0.046, WT=38 and KO=38 neurons, N=3 biological replicates). Scale bar: 5 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. (N) Significantly increased ratio of Αβ1-42/ Αβ1-40 measured by ELISA in the media of cultured AP-2μ KO neurons compared to the WT set to 100% (KO:124.57±10.97%, p=0.037, N=6 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. Accumulation of intracellular Aβ1−42 in neurons expressing the AP2-α−WT (WT: 1.14±0.18 , WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts). Accumulation of intracellular Aβ1−42 in neurons overexpressing the AP2-α-Mut comparing to neurons expressing the AP2-α−WT (WT: 1.14±0.18, Mut: 2.06±0.29, p=0.011, WT=28 and Mut=33 neurons, N=4 biological replicates). Scale bars: 20µm, 2µm (inserts). 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. Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down (WTScr: 2.76±0.20 WTshBACE1: 2.52±0.12 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm. Accumulation of intracellular Aβ1-42 is rescued upon BACE1 knock-down in AP-2µ KO neurons (WTScr: 2.76±0.20, KOScr: 3.94±0.21; WTshBACE1: 2.52±0.12; KOshBACE1: 2.74±0.21; pWTScr vs pKOScr=0.000, pKOscr vs pKOshBACE1=000; pWTscr vs pWTshBACE1=0.826; pWTshBACE1 vs pKOshBACE1=0.863; 27 to 39 neurons per condition, N=3 biological replicates). Scale bar: 10µm. Data information: All graphs show mean ± SEM, statistical analysis was performed by two-way ANOVA in (T) n.s.-non-significant. * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001. (U, V) Levels of intracellular Aβ1−42 are significantly upregulated in neurons overexpressing the HA-BACE1-LL/AA-GFP comparing to neurons expressing the HA-BACE1-GFP (BACE1: 0.78±0.90, BACE1-LL-AA: 1.99±0.33, p=000, 28-29 neurons per condition, N=3 biological replicates). Scale bar, 50µm. 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.