FIGURE 1:

mTOR inhibition decreases Rab11 mobility in dendritic spines. (A) Representative images of neurons transfected, using Lipofectamine 2000, at ∼ 3 wk in culture, with plasmid encoding EGFP (arrowheads) stained with Hoechst33258. The image shows healthy, nonshrunk nuclei of transfected cells. (B) Diagram of treatment and imaging for experiments in Figure 1. Neurons were recorded for 1 min (preincubation recording; pre-30′), then incubated for 30 min to control potential phototoxicity or overheating in the microscope stage. Then, neurons were rerecorded (0′) and incubated for 20 min without (ctrl 20′) or with 300 nM mTOR inhibitor INK128 (INK128 20′) for the final recording. (C) Representative time-lapse images of dendrites of rat hippocampal neurons that were cotransfected on DIV22 with plasmids encoding GFP-Rab11a and RFP and treated the next day with INK128 (300 nM). The figure shows Rab11a vesicles (green), RFP (magenta), an overlay with GFP-Rab11a (fire pseudocolor), and the outline based on the RFP channel (hand-drawn ROI). Arrows indicate dendritic spines with motile vesicles. Imaging occurred ∼17–24 h after transfection. Neurons were imaged three times, always for 1 min, at the baseline (unpublished data, in D and E; INK 128 pre-30′; see panels I and K), 30 min later just before treatment (INK128 0′) and after 20′ incubation with INK128 (INK128 20′). Control neurons (unpublished data in C) were imaged as described above without any treatment (ctrl pre-30′, ctrl 0′, and ctrl 20′, see H and J). Scale bar = 2.5 µm. (D) Western blot analysis of INK128 treatment effects on caspase 3 (casp 3) cleavage in cultured neurons. Primary hippocampal rat neurons (DIV21) were treated with INK128 (300 nM) for 20 min. Extracts were blotted against casp 3 with tubulin as a loading control. (E–G) Western blot analysis of mTORC1 and mTORC2 inhibition by INK128. The same extracts as in (D) were blotted against phosphorylated (Ser235/S236, P-S6) and total S6 and phosphorylated (Ser473, P-Akt) and total Akt to evaluate mTORC1 and mTORC2 suppression, respectively, with tubulin as a loading control. Data are presented as mean gray values for each phosphorylated protein normalized to total protein. n = 6 both for ctrl and INK128. ***p ≤ 0.001 (one-sample t test) (H) Percentage of Rab11a-positive spines in control neurons. (I) Percentage of Rab11a-positive spines in INK128-treated neurons. (J) Percentage of spines with mobile Rab11a in control neurons. (K) Percentage of spines with mobile Rab11a in INK128-treated neurons. The data are presented as a mean percentage of Rab11a positive spines normalized to all spines (H and I) or the mean percentage of Rab11a positive spines with mobile vesicles normalized to all Rab11a positive spines for all analyzed cells (J and K). n = 11 cells for treated and untreated neurons from four independent experiments. *p ≤ 0.05, **p ≤ 0.01 (repeated-measures ANOVA followed by Tukey’s post hoc test, all columns were compared with all columns).