Research Paper Volume 4, Issue 3 pp 187—201

Figure 3. EPO oversees Wnt1, mTOR and p70S6K and the PI 3-K/Akt1 pathway to protect microglia during Aβ exposure.

(A) Microglial protein extracts (50 μg/lane) were immunoblotted with phosphorylated mTOR (p-mTOR, (Ser²⁴⁴⁸)) and phosphorylated p70S6K (p-p70S6K, Thr³⁸⁹)) antibodies at 6, 12, and 24 hours following exposure to Aβ (10 μM). Aβ resulted in a slight increase in the expression of p-mTOR and p-p70S6K. In contrast, EPO (10 ng/ml) with a 1 hour pretreatment significantly increased the expression of p-mTOR and p-p70S6K during Aβ exposure (*P <0.01 vs. Control; †P<0.01 vs. Aβ of corresponding exposure time). In all cases, each data point represents the mean and SEM from 3 experiments. (B) Gene reduction ofWnt1was performed with transfection of Wnt1 siRNA prior to Aβ (10 μM) exposure in microglia. Expression of p-mTOR and p-p70S6K were determined at 6 hours following Aβ exposure. EPO (10 ng/ml) or Wnt1 (100 ng/ml) applied 1 hour prior to Aβ exposure significantly increased the expression of p-mTOR and p-p70S6K. Transfection with Wnt1 siRNA significantly reduced the expression of p-mTOR and p-p70S6K following a 6 hour period of Aβ exposure and during EPO (10 ng/ml) administration with Aβ exposure. Non-specific scrambled siRNA did not alter the expression of p-mTOR and p-p70S6K during Aβ exposure (*P<0.01 vs. Aβ; ^†P<0.01 vs. EPO/Aβ). (C and D) EPO (10 ng/ml), Wnt1 (100 ng/ml), or combined EPO with Wnt1 were applied to microglial cultures 1 hour prior to Aβ(10 μM) exposure and p-mTOR and p-p70S6K expression were determined 6 hours following Aβ exposure. EPO, Wnt1, or combined EPO with Wnt1 administration significantly increased the expression of p-mTOR and p-p70S6K to a similar levels 6 hours following Aβ exposure. Yet, combined application of the PI 3-K inhibitor LY294002 (10 μM, given 1.5 hours prior to Aβ) with EPO, Wnt1, or combined EPO with Wnt1 resulted in a significant decrease in expression of p-mTOR and p-p70S6K (*P<0.01 vs. Aβ; ^†P<0.01 vs. EPO/Aβ, Wnt1/Aβ, or EPO/Wnt1/Aβ). (E and F) EPO (10 ng/ml), Wnt1 (100 ng/ml), or combined EPO with Wnt1 were applied to microglial cultures 1 hour prior to Aβ exposure and cell survival was determined by the using trypan blue dye exclusion method 24 hours later. Representative pictures (E) and quantitative results (F) indicated that EPO, Wnt1, or combined EPO with Wnt1 application significantly reduced trypan blue staining and increased cell survival to a similar level following Aβ(10 μM) exposure. Application of the mTOR specific inhibitors rapamycin (Rapa, 50 nM) or KU 0063794 (KU, 100 nM) 1.5 hours prior to Aβ administration prevented EPO, Wnt1, or combined EPO with Wnt1 to foster cell survival and resulted in an increased staining of trypan blue and a decrease in cell survival in microglia (*P<0.01 vs. Aβ; †P<0.01 vs. EPO/Aβ, Wnt1/Aβ, or EPO/Wnt1/Aβ). Each data point represents the mean and SEM from 6 experiments.