FIGURE 11:

Depletion of AP-1γ in mouse primary cortical neurons results in increased APP processing. (A) E16 primary mouse cortical neurons were transduced at DIV3 with either AP-1γ-shRNA1 or AP-1γ-shRNA2 lentivirus (LV) for 96 h and treated with DMSO (carrier control), 2 μM DAPT, and/or 2 μM BACE1 inhibitor C3 in the last 16–20 h of transduction. Neurons were lysed in RIPA buffer and cell extracts (10 μg) subjected to SDS–PAGE, as described in Materials and Methods. Proteins transferred onto PVDF membrane were probed with mouse antibodies to either AP-1γ or α-tubulin. Proteins transferred onto nitrocellulose membrane were probed with rabbit anti-APP (Y188) antibodies to C99*/C83**. (B–D) Bar graph representing percentage of AP-1 depletion (B) and fold change of β-CTF/C99 (C) and α-CTF/C83 (D) in AP-1γ shRNA1 or AP-1γ shRNA2 lentiviral transduction compared with nontransduced controls. Levels of α-CTF/C83 and β-CTF/C99 were normalized to α-tubulin. Data are represented as the mean ± SD of three independent experiments and analyzed by one-way ANOVA using Tukey’s test. **p < 0.01, *p < 0.05, and n.s. = not significant. (E, F) Primary mouse cortical neurons were transduced at DIV3 with either AP-1γ-shRNA1 or AP-1γ-shRNA2 lentivirus, and 80 h after transduction the medium was changed and spent medium containing secreted Aβ was collected 16 h later and analyzed with a sandwich ELISA specific for either Aβ40 (E) or Aβ42 (F). The Aβ40 or Aβ42 levels for each sample were normalized against the total cellular protein. Data are represented as the mean ± SEM of three independent experiments and analyzed by one-way ANOVA using Tukey’s test. ***p < 0.001, **p < 0.01, and *p < 0.05. (G) Model of anterograde transport pathway of newly synthesized APP and BACE1. Both BACE1 (green bars) and APP (red bars) are synthesized in the ER and transported through the Golgi. Upon arrival at the TGN, APP and BACE1 are sorted for TGN export by distinct transport machinery. BACE1 is transported from the TGN directly to the PM via an AP-1-/Arf1-/Arf4-dependent pathway. APP is transported via an AP-4-/Arl5b-dependent pathway from the TGN directly to the early endosomes and then to either the PM or the late endosomes/lysosomes.