(a) Neuronal PC12 cells were transfected with either eGFP/pcDNA3 (control) or eGFP/pcDNA3-Wild-Type mTOR (WT) and after 48 hours, were treated with or without 50 μM 6-OHDA. Replicate cultures were pre-treated for one hour with 1 μM rapamycin as indicated prior to 6-OHDA treatment and maintained with rapamycin for the duration of the experiment. One day later, transfected viable cells were scored 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 cells transfected with eGFP/pcDNA3 empty vector; ** p < 0.01 compared with cells transfected with eGFP/pcDNA3 empty vector exposed to 50μM 6OHDA. (b,c,d) Neuronal PC12 cells were transfected with eGFP/pcDNA3 or eGFP/pcDNA3 WT mTOR vector. After 48 hours, cultures were treated with 50 μM 6-OHDA for 24 hours. Cultures were then immunostained with antibodies to P-(Ser2448)-mTOR, P-(Ser235/236)S6 and P-(Thr37/46) 4EBP1, respectively, along with antiserum to GFP to visualize transfected GFP+ cells. Proportions of transfected cells positive for phospho-mTOR, phospho-S6 or phospho-4EBP1 were scored under fluorescence microscopy. Values represent means ± SEM for three independent experiments done in triplicate. * p < 0.05 compared with cells transfected with pcDNA3 empty vector treated with 6-OHDA. (e) Neuronal PC12 cells were exposed to 250 nM or 1 μM Torin 1 for 5 hours. Cell extracts were then collected and subjected to SDS-PAGE and Western immunoblotting. Membranes were probed with the indicated immunoreagents and reprobed with an antibody for total Akt, as a loading control.

(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.

(a) Mice were injected with either vehicle, rapamycin, MPTP or rapamycin + MPTP, as described in Methods for the acute regimen, and sacrificed after 24 hours. Ventral midbrains were dissected, homogenized in lysis buffer and the homogenates subjected to SDS-PAGE and Western immunoblotting. Membranes were probed with RTP801 antibody and reprobed with an antibody for total ERK1/2 as a loading control. Film resulting from the immunoblot was scanned and relative densities of RTP801 bands were normalized in each case to the densities of the corresponding ERK1/2 signals. Values (in arbitrary units) for RTP801 are expressed as mean ± SEM for at least three mice per condition. *p<0.01 vs mice injected with vehicle; ^ψp<0.01 vs mice injected with MPTP. (b) Mice subjected to the acute regimen as indicated were sacrificed 7 days after the last MPTP injection. Midbrains were cryo-sectioned, immunostained with tyrosine hydroxylase antibody and subjected to Nissl staining. Unbiased stereology was used to determine the total numbers of TH positive neurons in the substantia nigra. Results represent mean ± SEM of total number of TH-positive cells per substantia nigra (right and left) for at least 7 mice per each condition. **p<0.001 vs mice injected with vehicle; ++p<0.01 vs mice injected with rapamycin+MPTP. Lower panel shows representative micrographs of SN stained with TH-antibody in comparable sections for each condition. (c) Mice subjected to the subacute MPTP regimen as indicated were sacrificed two days after the last injection. Midbrains were cryo-sectioned, immunostained lightly with tyrosine hydroxylase antibody and subjected to Nissl staining to visualize condensed nuclei (with fragmented chromatin). Results represent mean ± SEM of total number of apoptotic nuclei per midbrain for at least 4 mice per each condition. ***p<0.001 vs mice injected with MPTP alone.

(a) Neuronal PC12 cells were pretreated with or without 0.1 or 1 μM rapamycin (upper panel) or with 20 nM rapamycin (lower panel) and exposed to 100 μM 6-OHDA for 8 hours. Cell extracts were subjected to SDS-PAGE and Western immunoblotting. Membranes were probed with the indicated immunoreagents and reprobed with antibodies for ERK1/2 or total Akt as loading controls. Right-hand panels show quantification (means ± SEM) from 3 or more independent experiments. RTP801 relative expression (normalized to ERK1/2): *p< 0.05 vs untreated control (ct) cultures; ++p< 0.01, +++p< 0.001 vs 6-OHDA treated cultures. P-S6K relative expression (normalized to ERK1/2): **p< 0.01 vs untreated control cultures; +p< 0.05 vs 6-OHDA-treated cultures.
(b) Neuronal PC12 cells were pretreated with or without 1 μM rapamycin or 1 μM FK506 and then exposed to 100 μM 6-OHDA for 8 hours. Cell extracts were subjected to SDS-PAGE and Western immunoblotting. Membranes were probed with the indicated immunoreagents and reprobed with antibodies for total S6K and ERK1. The vertical white line between lanes in figure b lower panel, indicates elimination of irrelevant intervening lanes in the blot. (c) Neuronal PC12 cells were pretreated with or without 1 μM rapamycin and then exposed to 100 μM 6-OHDA or 500 μM MPP+ for 8 hours. Cell extracts were subjected to SDS-PAGE and Western immunoblotting. Membranes were probed with the indicated immunoreagents and reprobed with an antibody for total ERK1 as loading control. Right-hand panel shows quantification (means ± SEM; normalized to ERK1/2) from 3 or more independent experiments. RTP801 relative expression: **p< 0.01, *p<0.05 vs untreated control cultures; ++p< 0.01 vs 6-OHDA treated cultures; ##p< 0.01 vs MPP+ treated cultures. (d) Sympathetic neuron cultures were pretreated with or without 1 μM rapamycin and then exposed to 10 μM 6-OHDA for 16 hours. Cell extracts were analyzed by Western immunoblotting with the indicated immunoreagents. Right-hand panel shows quantification (means ± SEM; normalized to ERK1/2) from 3 or more independent experiments. RTP801 relative expression: *p< 0.05 vs untreated control (ctl) cultures. The vertical white line between lanes indicates elimination of irrelevant intervening lanes in the blot.

(a) Neuronal PC12 cells were pretreated with 1 μM rapamycin and then exposed to 1 mM MPP+ for 8 hours. RNA was extracted and RT-PCR was performed to quantify the number of copies of cDNA coding for RTP801 under the indicated conditions. Values represent mean ± SEM of at least four independent experiments. *p< 0.05 vs control (Ctl); **p< 0.01 vs control (Ctl). (b) Neuronal PC12 cells were pretreated with 1 μM rapamycin and then exposed to 1 mM MPP+ for 8 hours. Then, they were treated with or without 1 μM cycloheximide for 10 or 60 minutes. Cell extracts were collected immediately after and subjected to SDS-PAGE and Western immunoblotting. Membranes were probed with the indicated immunoreagents and reprobed with antibodies for total ERK1 and Akt. Densitometry values represent mean ± SEM of at least three independent experiments. * p< 0.05 vs control (-) and + p< 0.05 vs MPP+ alone. (c) Neuronal PC12 cells were pretreated with or without 1 μM rapamycin and exposed to 100 μM 6-OHDA for 16 hours. Cell extracts were analyzed by SDS-PAGE and Western immunoblotting. Membranes were probed with anti-phospho-Thr308-Akt, anti-phospho-Ser473-Akt and anti-RTP801 antibodies. Membranes were reprobed with antibody against total Akt as loading control. Films were scanned and densitometries (ImageJ, NIH, USA) were expressed as arbitrary units versus total Akt loading control for the two phospho-Akt residues (d) and RTP801 (e). Values represent mean ± SEM of at least three independent experiments in each condition. #p< 0.05 vs phospho-Ser473-Akt in control extracts (Ct); *p< 0.05 vs phospho-Thr308-Akt in control extracts (Ct); +p< 0.05 vs phospho-Thr308-Akt in 100 μM 6OHDA treated cells.

(a) Under basal conditions, RTP801 gene and protein expression are low. mTOR activity is unaffected and promotes rapamycin-sensitive and -insensitive translation as well as phosphorylation of Akt at Ser473 and Thr308. Cell survival is favored. (b) In presence of Torin1, all mTOR activities including Akt phosphorylation at Ser473 and Thr308 and rapamcyin-sensitive and -insensitive translation are blocked. RTP801 expression is low. Cell death is favored due to inhibition of Akt phosphorylation/activation. (c) In presence of PD toxins, RTP801 gene and protein expression are induced. This leads to RTP801-dependent inhibition of mTOR activation and consequently to inhibition of Akt phosphorylation at Ser473 and Thr308. Cell death is favored. While mTOR inhibition will affect RTP801 translation, the increased half-life of RTP801 protein in presence of PD toxins and mutual feedback relationship between mTOR activity and RTP801 translation likely account for the sustained elevation of RTP801 levels in presence of the toxins. (d). Rapamycin does not affect induction of RTP801 transcripts by PD toxins, but blocks mTOR-dependent RTP801 protein synthesis (most likely through inhibition of mTORC1-dependent p70S6 kinase activation) and phosphorylation of Akt at Ser473 (most likely through inhibition of mTORC2). Other activities of mTOR are left intact such as cap-dependent translation. Phosphorylation of Akt at Thr308 is also spared, leading to cell survival. This sparing of Thr308 phosphorylation may reflect inhibition by rapamycin of m-TOR-dependent p70S6 kinase activation and of RTP801 synthesis. In addition, inhibition of p70S6 kinase activity may enhance Akt Thr308 phosphorylation by leading to stabilization of IRS-1 and to enhanced PI3K activity.