FIGURE 3. Rapamycin protects cultured neuronal PC12 cells and sympathetic neurons from PD mimetics.

(a) Neuronal PC12 cells were pretreated with the indicated concentrations of rapamycin or FK506 for one hour and then exposed to 100 μM 6-OHDA. Survival was measured 24 hours later. Values are expressed as mean ± SEM of at least three independent experiments in triplicate in each condition. **p <0.01 and ***p<0.001 compared with untreated cultures exposed to 6-OHDA. (b) Neuronal PC12 cells were pretreated with 1 μM rapamycin for an hour and then exposed to either no treatment (control), 100 μM 6-OHDA or 200 μM MPP+ in the continued presence of rapamycin. Survival was measured 24 hours later. Values are expressed as mean ± SEM of at least three independent experiments in triplicate in each condition. +++p < 0.001 compared with untreated cultures; ***p<0.001 compared with cultures treated with 6-OHDA alone; *p<0.05 compared with cultures treated with MPP+ alone. (c) Images of neuronal PC12 cells cultures treated with or without rapamycin and then exposed to the indicated PD mimetics. Note in the right-hand panel that rapamycin diminishes neurite loss in neuronal PC12 cell cultures induced by PD mimetics. *p<0.05 and ***p<0.001 compared with untreated control cultures; ++ p<0.01 compared with 6OHDA treated cultures alone; # p<0.05 compared with MPP+ alone. (d). Sympathetic neurons at 7 days in vitro were pretreated for an hour with 20 nM, 0.1 μM or 1 μM rapamycin and then exposed to 15 μM 6-OHDA. After 24 hours, cultures were fixed and stained with nuclear dye Hoechst 33342. Surviving neurons (judged by the lack of chromatin condensation) were strip-counted under fluorescence microscopy. Values are expressed as mean ± SEM of at least three independent experiments in triplicate in each condition. **p < 0.01 compared with untreated cultures exposed to 6-OHDA.