Impaired Function of HDAC6 Slows Down Axonal Growth and Interferes with Axon Initial Segment Development
Figure 5
HDAC6 inhibition or suppression interfere the concentration of ankyrinG and voltage dependent sodium channels at the axon initial segment.
(A) Control or TSA treated 6 DIV hippocampal neurons stained with the somatodendritic marker, MAP2 (red), and the AIS markers, ankyrinG or PanNaCh (green). Control neurons concentrate ankyrinG and sodium channels in the AIS, while in TSA treated neurons ankyrinG staining is distributed all along the axon, and voltage gated sodium channels staining is very low or lost. Arrow head indicate the position of the axon. Scale bar = 50 µm. (B) Percentage of neurons with ankyrinG or voltage gated sodium channels concentrated in the AIS. Data represent the mean ± SEM of 3 independent experiments (500 neurons/experimental condition and experiment). ***p<0.001, ****p<0.0001, paired t-test. (C) Western-blot shows the expression levels of the α-subunit of voltage gated sodium channels in control and 100 nM TSA treated neurons compared to β-actin expression levels. (D) 6 DIV hippocampal neurons treated with DMSO, tubacin or niltubacin from 3 DIV to 6 DIV, and then stained for MAP2 (red) and ankyrinG or sodium channels (green). (E) Percentage of neurons that show a localized concentration of ankyrinG or sodium channels at the axon initial segment. The graphs represent the mean ± SEM of 3 independent experiments (600 neurons/experimental condition and experiment). *p<0.05, paired t-test. Scale bar = 100 µm. (F) 3 DIV hippocampal neurons nucleofected with an HDAC6 shRNA plasmid or the control plasmid. After 3 days in culture, neurons were stained for ankyrinG and the neuronal morphology was distinguished by GFP fluorescence. Rectangles indicate the magnified zone shown in lower panels. Lower panels show a magnification of both neurons along the axon with ankyrinG concentrated at the initial region of the axon (control neurons, pGFP-V-RS) or along the axon (pGFP-V-RS-shRNA-HDAC6 nucleofected neurons). Scale bar = 100 µm. (G) 3 DIV hippocampal neurons nucleofected as in E and stained with an anti-PanNaCh antibody. Square inserts show a magnification of both neurons at the level of the AIS. Scale bar = 100 µm. (H, I) Percentage of GFP, scramble shRNA or HDAC6 shRNAs nucleofected neurons that concentrate ankyrinG (G) or sodium channels (H) in the AIS after 3 days in culture. Data are the mean ± SEM of 3 independent experiments (100 neurons/experimental condition and experiment). * p<0.05, **p<0.01, t-test.