Figure 1.
Rapamycin suppresses mTOR phosphorylation at Ser-2448 in a dose dependent manner.
Primary cortical neurons were treated under normoxic conditions with various concentrations of rapamycin (0 nM – 20 nM). (A,B) Rapamycin caused a dose-dependent decrease in phosphorylated mTOR at Ser-2448, and subsequently caused a dose-dependent decrease in phosphorylated p70S6K. (C,D) Rapamycin at 20 nM caused a small decrease in phosphorylated mTOR at Ser-2481. The lower concentrations of rapamycin (2 nM – 10 nM) increased the phosphorylation of Akt, whereas 20 nM of rapamycin caused a decrease in phosphorylated Akt. (A,B) Representative immunoblots. (B,D) Quantitative analysis is shown as percent control for all phospho-proteins normalized to total protein. Data is represented as the mean ± s.e.m. (n = 4). *p<0.05 vs. 0 nM, #p<0.05 10 nM vs. 20 nM.
Figure 2.
Rapamycin treatment increases cell survival following OGD.
Primary cortical neurons were subjected to 1 hour OGD and then treated with rapamycin (20 nM) or vehicle (0.01% EtOH) for 90 m and 24 h. Cultures were immunostained with Hoechst (blue, nucleus stain) and MAP-2 (red, neuronal marker). MAP-2-positive cells were counted in 5 random fields on each of three cover slips per condition. Rapamycin significantly improved cell survival at both 90 m and 24 h following OGD when compared to vehicle. Data is represented as the mean ± s.e.m. (n = 3). *p<0.05 rapamycin vs. vehicle.
Figure 3.
Rapamycin has no effect on caspase-3 in ischemic neurons.
Primary cortical neurons were subjected to 1 h OGD and then treated with rapamycin (20 nM) or vehicle (0.01% EtOH) for multiple time points (3 h – 24 h). OGD for 1 h caused an increase in caspase-3 cleavage that peaked after 6 h. However, rapamycin had no effect on caspase-3 cleavage when compared to vehicle. (A) Representative immunoblots. (B) Quantitative analysis is shown as percent control for cleaved-caspase-3 normalized to caspase-3. Data is represented as the mean ± s.e.m. (n = 3).
Figure 4.
Rapamycin decreases phosphorylated mTOR following OGD.
Primary cortical neurons were subjected to 1 hour OGD and then treated with rapamycin (20 nM) or vehicle (0.01% EtOH) for 24 h. (A) Cells were immunostained with phospho-mTOR Ser-2448 (green), Hoechst (blue) and MAP-2 (red). Under control conditions, Ser-2448 is mainly localized in the cytoplasmic domain. Under OGD conditions, rapamycin decreased the amount of Ser-2448 immunoreactivity. (B) Cells were immunostained with phospho-mTOR Ser-2481 (green), Hoechst (blue) and MAP-2 (red). Under control conditions, Ser-2481 is seen throughout the cell body, including the cytoplasm, nucleus and cell extensions. Under OGD conditions, rapamycin decreased the amount of Ser-2481 immunoreactivity. Images are representative figures (n = 3).
Figure 5.
Following OGD mTOR phosphorylation is reversed by rapamycin treatment.
Primary cortical neurons were subjected to 1 h OGD and then treated with rapamycin (20 nM) or vehicle (0.01% EtOH) for multiple time points (1.5 h–24 h). (A,B) Following OGD, rapamycin significantly decreased the phosphorylation of mTOR at both Ser-2448 and Ser-2481 compared to vehicle for all time points. (C,D) Following OGD, rapamycin significantly decreased the phosphorylation of p70S6K, the downstream target of mTORC1. However, rapamycin only caused a small decrease in phorphorylated AKT after 24 h. Rapamycin had no effect at any other time point. (A-D) Quantitative analysis is shown as percent control for all phospho-proteins normalized to total protein. Data is represented as the mean ± s.e.m. (n = 3). *p<0.05 vs. vehicle. (E,F) Representative immunoblots.