Figure 3. STK11-PRKAA1 activates autophagic degradation of Aβ fibrils by microglia. (A) The BV2 microglial cell line and primary mouse microglia were treated with Aβ fibrils (1 μM) for 2 h. Whole cell lysates were collected and analyzed by immunoblotting with indicated antibodies. The levels of p-STK11 and p-PRKAA1 (Thr172) were increased in fAβ-treated microglia. (B) The BV2 microglial cell line was transfected with the indicated siRNAs and Prkaa1 silent-mutant and treated with Aβ fibrils (1 μM) for 24 h. Immunoblots of 6E10 and ACTB in BV2 that were transfected with the indicated siRNAs and treated with Aβ fibrils (1 μM) for 24 h. High levels of Aβ were observed in siPrkaa1-transfected microglia compared with siCTL. (C) BV2 microglial cells were preincubated with AICAR, metformin, or compound C for 30 min before Aβ fibrils (1 μM) were added. Whole cell lysates were collected 24 h later, and then the lysates were analyzed by immunoblotting for 6E10 and ACTB (loading control). Lower levels of Aβ were observed in AICAR- and metformin-treated microglia, but higher levels of Aβ were observed in compound C-treated microglia. (D) The BV2 microglial cell line and primary mouse microglia were preincubated with AICAR, metformin, or compound C for 30 min before FITC-labeled Aβ fibrils (1 μM) were added. The cells were observed 24 h later. Representative images show that FITC-labeled Aβ was lower in AICAR- and metformin-treated microglia but higher in compound C-treated cells. Scale bar: 10 μm. (E and F) Immunoblots of primary microglia cell lysates demonstrating that the decrease in Aβ by AICAR or metformin was abolished in siMap1lc3b- and siAtg7 transfected microglia. The bar graphs show the densitometric quantification of the immunoblot bands. Each graph shows the band densities of the immunoreactive proteins as a percentage of the indicated group. Data are presented as the means ± SEM of 3 independent experiments and were analyzed using the Student t test. **P < 0.01, **P < 0.005 vs. control.