Figure 3.
Figure 3. Altered neuronal polarity correlates with changed MT stability. (A and B) Rat hippocampal neurons grown in the presence of the GSK-3β inhibitor SB 415286 for 72 (A) and 24 h (B). Stage 3 neurons have formed multiple axons that feature an increased MT stability (A, arrows) compared with minor neurites (A, arrowhead). A rise in MT acetylation in several minor neurites (B, arrowheads with asterisk) precedes the formation of multiple axons in morphologically still unpolarized stage 2 neurons treated with GSK-3β inhibitor. Arrows, axons; arrowhead, minor neurites. Bars, 20 μm. (C) Ratio quantification of fluorescence intensities of acetylated and tyrosinated α-tubulin in MTs of unpolarized stage 2 neurons. The normalized average ratio of the five longest neurites is shown. Approximately 5 h after treatment with SB 415286, stage 2 neurons show a trend toward increased MT acetylation (23.0 ± 10.6%), which indicates a rise in MT stability. The increase is significant after ∼8 h (47.4 ± 8.7%; mean ± SEM; n > 50 neurons per condition and time point from three independent experiments; *, P < 0.05 by t test). (D) Rat hippocampal neurons (3 DIV) treated with the GSK-3β inhibitor SB 415286 (10–20 μM treatment 6–8 h after plating) formed 2.1 ± 0.1 axons on average. These supernumerary axons show an increased ratio of acetylated to tyrosinated α-tubulin equal (P > 0.35 by t test) to that of the single axon of stage 3 control neurons (treatment with 0.04% DMSO; mean ± SEM; n > 35 neurons from three independent experiments). (E and F) Hippocampal neurons (3 DIV) derived from mice deficient for SAD A and B kinase show disturbed polarity and lack a defined axon. Instead, SAD A/B knockout neurons form multiple processes of similar length and uniform tubulin acetylation levels (see G), yet a high cell-to-cell variability. Bars, 20 μm. (G) Ratio quantification of fluorescence intensities of acetylated and tyrosinated α-tubulin in MTs. Processes of SAD A−/−B−/− neurons are short of the specific enrichment of acetylated MTs in one process found in wild-type as well as littermate control neurons (SAD A+/+B+/−; mean ± SEM; n = 66 and 27 neurons from three independent experiments for SAD A−/− SAD B−/− and control, respectively). Note that the acetylation/tyrosination ratio varies slightly in control cells between species (rat vs. mouse; D and G). (H) Ratio of the acetylation/tyrosination ratios of the longest versus second longest process per cell for SAD A−/−B−/− and control neurons (mean ± SEM; ***, P < 0.001 by t test).

Altered neuronal polarity correlates with changed MT stability. (A and B) Rat hippocampal neurons grown in the presence of the GSK-3β inhibitor SB 415286 for 72 (A) and 24 h (B). Stage 3 neurons have formed multiple axons that feature an increased MT stability (A, arrows) compared with minor neurites (A, arrowhead). A rise in MT acetylation in several minor neurites (B, arrowheads with asterisk) precedes the formation of multiple axons in morphologically still unpolarized stage 2 neurons treated with GSK-3β inhibitor. Arrows, axons; arrowhead, minor neurites. Bars, 20 μm. (C) Ratio quantification of fluorescence intensities of acetylated and tyrosinated α-tubulin in MTs of unpolarized stage 2 neurons. The normalized average ratio of the five longest neurites is shown. Approximately 5 h after treatment with SB 415286, stage 2 neurons show a trend toward increased MT acetylation (23.0 ± 10.6%), which indicates a rise in MT stability. The increase is significant after ∼8 h (47.4 ± 8.7%; mean ± SEM; n > 50 neurons per condition and time point from three independent experiments; *, P < 0.05 by t test). (D) Rat hippocampal neurons (3 DIV) treated with the GSK-3β inhibitor SB 415286 (10–20 μM treatment 6–8 h after plating) formed 2.1 ± 0.1 axons on average. These supernumerary axons show an increased ratio of acetylated to tyrosinated α-tubulin equal (P > 0.35 by t test) to that of the single axon of stage 3 control neurons (treatment with 0.04% DMSO; mean ± SEM; n > 35 neurons from three independent experiments). (E and F) Hippocampal neurons (3 DIV) derived from mice deficient for SAD A and B kinase show disturbed polarity and lack a defined axon. Instead, SAD A/B knockout neurons form multiple processes of similar length and uniform tubulin acetylation levels (see G), yet a high cell-to-cell variability. Bars, 20 μm. (G) Ratio quantification of fluorescence intensities of acetylated and tyrosinated α-tubulin in MTs. Processes of SAD A−/−B−/− neurons are short of the specific enrichment of acetylated MTs in one process found in wild-type as well as littermate control neurons (SAD A+/+B+/−; mean ± SEM; n = 66 and 27 neurons from three independent experiments for SAD A−/− SAD B−/− and control, respectively). Note that the acetylation/tyrosination ratio varies slightly in control cells between species (rat vs. mouse; D and G). (H) Ratio of the acetylation/tyrosination ratios of the longest versus second longest process per cell for SAD A−/−B−/− and control neurons (mean ± SEM; ***, P < 0.001 by t test).

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