Figure 1.
NAP affects the α-tubulin tyrosination cycle.
Glu-MT and Tyr-MT fluorescence levels in cultured cells after exposure for 2 hrs to different drug concentrations. Cells were permeabilized during fixation to remove non-polymerized tubulin subunits, therefore only tubulin incorporated into MT was assessed. (a) Concentration response analysis of fluorescence intensity units (FAU) normalized to Hoechst signal in paclitaxel-treated PC12 cells evaluated by “In cell western”, microplate reader method (Glu-MT, ANOVA, p<0.0001, n = 9 per treatment), (Tyr-MT, ANOVA, p<0.0001, n = 9 per treatment). The dotted line indicates the paclitaxel concentration, 5 µM, used in the following experiments as a control. (b) Concentration-response analysis of fluorescence intensity units (FAU) normalized to Hoechst signal in NAP- (10−18M - 10−6M) or vehicle-treated cells. PC12 cells were evaluated by “In cell western”, microplate reader method. (Glu-MT, ANOVA, p<0.0001, n = 9 per treatment), (Tyr-MT, ANOVA, p = 0.0139, n = 9 per treatment) (c) Concentration response analysis of fluorescence intensity ratio levels (Tyr-MT/Glu-MT) in PC12 cells treated with NAP (10−18M - 10−6M) or vehicle, evaluated by image analysis of confocal images. The control compound was paclitaxel, 5 µM, (ANOVA, p = 0.0001, n>34 cells per treatment). The fluorescence intensities of both Tyr-MT and Glu-MT in the colchicine-treated cells were below the threshold level of measurements (see d). (d) Comparison of the effect on Glu-MT and Tyr-MT and integrity of the MT network. Control compounds were paclitaxel, 5 µM and colchicine, 2 µM (bottom row). PC12 stained for Tyr-MT (red), Glu-MT (green), Hoechst (blue). Bars: 10 µm. (e) Concentration response analysis of fluorescence intensity ratio levels (Tyr-MT/Glu-MT) in rat cerebral cortical astrocytes evaluated by image analysis of confocal images. (ANOVA, p<0.0001, n = 27). The fluorescence intensities of both Tyr-MT and Glu-MT in the colchicine-treated cells were below the threshold level of measurements (see f). (f) Comparison of the effect on Glu-MT and Tyr-MT and integrity of the microtubular network. Control compounds were paclitaxel, 5 µM and colchicine, 2 µM (bottom row). Rat cerebral cortical astrocytes stained for Tyr-MT (red), Glu-MT (green), Hoechst (blue). Bars: 10 µm. (g) Concentration response analysis of fluorescence intensity ratio levels (Tyr-MT/Glu-MT) in NIH3T3 cells evaluated by image analysis of confocal images. (ANOVA, p = 0.9966, n = 18).
Figure 2.
NAP effect on the polymerized tubulin fraction in PC12 cells.
PC12 cells were treated for 2 hrs with either NAP (10−18M - 10−6M) or vehicle, and control compounds paclitaxel, 5 µM and colchicine, 2 µM. An additional time point (4 hrs.) was included as well for NAP (10−9M), vehicle and paclitaxel, 5 µM. (a) Cell lysates were separated into polymerized (P) or soluble (S) protein fractions. Aliquots of equal volume for each pair were resolved on adjacent lanes by SDS polyacrylamide gel electrophoresis, the blot probed with anti α-tubulin (2 hrs.) and with anti actin. The intensity of each band was quantified by densitometry and the percentage of polymerized MT was calculated by dividing the densitometric value of polymerized tubulin (P) by the total tubulin content (the sum of P plus S) in the 2 hr-incubation period and plotted as a line graph (ANOVA, p = 0.1868, n = 7). (b) The 4 hr-incubation samples shown in (a) were subjected to densitometry as above and the data plotted in bar graphs. Pairs of treatment were compared for each of the variables tubulin, actin (t-test p<0.05; ***p<0.005, n = 3).
Figure 3.
NAP enhances tau-microtubule interactions and microtubule polymerization under zinc intoxication.
(a) Zinc exposure (400 µM) resulted in PC12 cell death which was protected against by NAP treatment. Results of mitochondrial activity (MTS cell viability) are shown –100– is 100% survival – control w/o zinc treatment. (ANOVA, ***p<0.0001, n = 18/group, post hoc comparison were made in reference to vehicle+Zn treatment), upper panel. The lower panel shows a similar experiment with 5 µM paclitaxel (ANOVA, ***p<0.0001, n = 10/group, post hoc comparisons made in reference to vehicle, or to vehicle+Zn treatment). (b) Cell lysates were separated into polymerized (P) or soluble (S) protein fractions as in Figure 2. Aliquots of equal volumes for each pair were separated on adjacent lanes by SDS polyacrylamide gel electrophoresis, the blot was probed with antibodies recognizing α-tubulin, or total tau or actin and the percentage of the polymerized fraction was calculated for each ‘P’ and ‘S’ pair. (c) The intensity of each band was quantified by densitometry, and the percentage of the polymerized fraction was determined. (Actin, ANOVA, p = 0.4774, n = 6; Tubulin, ANOVA, p = 0.007, n = 6; TAU, ANOVA, p = 0.0022, n = 5, post hoc determinations were against the zinc-treated group for each of the variables).
Figure 4.
Treatment with NAP increases total polymerized α-tubulin network area in PC12 cells.
(a) Concentration response analysis of MT network area/cell as determined by the outer boundaries of fluorescently probed MT (monoclonal anti α-tubulin) in PC12 or NIH3T3 cells, evaluated by image analysis of confocal images. Cultured cells were exposed for 24 hr. to different concentrations of NAP (10−18M - 10−6M) and compared to vehicle-treated cells and paclitaxel, 5 µM treated cells. (PC12 - ANOVA, p = 0.0081, n = 87 cells per treatment; NIH3T3 - ANOVA, p = 0.3892, n = 90). Left side, Representative field-images of PC12 cells treated with paclitaxel with fluorescently probed MT. Bar: 10 µm (b) Representative field-images of PC12 cells with fluorescently probed MT (control and NAP-treated as indicated). Bars: 10 µm.
Figure 5.
NAP effect on Tyr-MT invasion to growth cones.
(a) Glu-MTs (green) do not invade the peripheral domain of the growth cone. NAP clearly affected Tyr-MT (red) invasion to the peripheral domain of the growth cone in terms of number and length of this invasions. Actin is labeled in blue (Coumarin Phalloidin) and cell membranes are stained in cyan (Dil). Bars: 7.5 µm. (b) Analysis of MT invasion - the total area in the image covered by actin and membrane dyed using DiI determined the growth cone area (T). From this determined area we subtracted the area occupied by Glu-MT (proximal growth cone area, P) which gave us our region of interest (ROI), as illustrated, with a representative picture and termed Atyr (area including Tyr-MT). (c). The percentage of the ROI penetrated by Tyr-MT was obtained, calculated and graphed, right hand side, (t-test, p<0.0001, n = 22/treatment group).
Figure 6.
NAP affects β3-tubulin expression levels in PC12 cells.
Protein expression levels of β3-tubulin were measured in PC12 cells maintained 8 days in culture. Comparisons were made after exposure to vehicle or different concentrations of NAP (10−18M - 10−6M). Control cultures were treated with medium only or with NGF (a) Concentration response analysis of protein expression levels were evaluated by western blotting of whole cell extracts with β3-tubulin immunoreactivity (top), and actin, control immunoreactivity (bottom). (b) Densitometric quantification of protein expression performed with ImageJ software. (ANOVA, p = 0.0009, n = 5, post hoc comparison are made in reference to vehicle treatment).