Figure 3. Enhancement of retromer function or autophagy rescues Aβ and tau phenotypes, while modulation of SMAD signaling regulates APOE levels in a SORL1-dependent manner.

(A–C) Line #1 iNs were treated with R33, a retromer stabilizer, at either 10 or 20 μM for 72 h and harvested at D21 of differentiation. A representative western blot of lysates from these SORL1 WT and KO iNs is shown in (A). Quantification shown of the protein expression of p-tau/tau (p202/205) (B) and APOE/GAPDH (C).

(D–F) iNs were treated with trehalose, a putative enhancer of autophagy, at 100 mM for 72 h and harvested at D21 of differentiation. A representative western blot of these lysate is shown in (D). Quantifications shown for p-tau/tau (p202/205) (E) and APOE/GAPDH (F).

(G–I) iNs were treated with 5 μM chloroquine (CQ) (to inhibit autophagic vesicle fusion with the lysosome) for 72 h and harvested at D21 of differentiation. A representative western blot of lysates is shown (G). Quantifications shown for LC3b-II/GAPDH (H) and APOE/GAPDH (I).

(J) SORL1 WT and KO iNs were treated with vehicle, TGF-β, or SB-431542 for 72 h. Lysates were collected, RNA purified, and qPCR performed for APOE and GAPDH (J, left) or else cells were lysed and western blotting was performed for APOE and GAPDH (J, right). For all quantifications in (B)–(J), data show mean ± SEM from three independent differentiations. For each round of differentiation, three replicates were included for each group. Values are normalized to WT iNs treated with vehicle. One-way ANOVA with Tukey’s multiple comparisons test.

(K) Schematic showing the intracellular life cycle of APOE, including a speculative model for the regulation of APOE by TGF-β signaling in neurons, with activation of TGF-β signaling resulting in repression of APOE transcription.