Figure 1.
Inhibition of HDAC6 by tubacin dramatically promotes mitochondrial movement in hippocampal neurons.
Kymographs (A, B and C) depicting mitochondrial motility were made from Movies S1, S2, S3, S4, S5, S6, and S7, S8, S9 respectively. In the experiment, a segment of axon was imaged continuously for 3 hours with a short break to allow for administration of drugs. Images were acquired at 10-second intervals. A. Treatment with tubacin. B. Treatment with TSA. C. Treatment with niltubacin. D, E and F. Quantification of the numbers of moving mitochondrial during the period of observation (n = 6). Only mitochondria that moved through the entire field of view were counted. G, H and I. Quantification of mean velocities of moving mitochondria (n = 5).
Figure 2.
Changes in tubulin acetylation and association of kinesin-1B with mitochondria occur following treatment with tubacin, TSA, or LiCl.
A. Western blot analysis of kinesin-1B and acetylated tubulin levels in total cell lysates from hippocampal neuronal cultures that were treated with tubacin (20 µM) or TSA (10 µM) for one hour. B. Western blot analysis of kinesin-1B and acetylated tubulin levels in total cell lysates from hippocampal neuronal cultures that were treated with niltubacin (20 µM) or LiCl (10 mM) for one hour. C. Quantification of Western blot results shown in A (n = 3). D. Quantification of Western blot results shown in B (n = 3). E. Western blot analysis of kinesin1-B and acetylated tubulin protein levels in a mitochondrial fraction isolated from hippocampal neuronal cultures that were treated with tubacin (20 µM) or TSA (10 µM) for one hour. F. Western blot analysis of kinesin1-B and acetylated tubulin protein levels in a mitochondrial fraction from hippocampal neuronal cultures that were treated with niltubacin (20 µM) or LiCl (10 mM) for one hour. G. Quantification of Western blot results shown in E (n = 3). H. Quantification of Western blot results shown in F (n = 3). Mitochondrial protein levels were normalized to voltage-dependent anion channel (VDAC) protein levels.
Figure 3.
GSK3β activity regulates both mitochondrial movement and tubulin acetylation.
Inhibition of GSK3β by LiCl or SB216763 promotes mitochondrial movement and increases levels of acetylated tubulin, whereas activation of GSK3β via inhibition of Akt decreases levels of acetylated tubulin in hippocampal neurons. Kymographs (A and B) of mitochondrial motility correspond to Movies S10, S11, S12 and S13, S14, S15 respectively. In the experiment, a segment of axon was imaged continuously for 3 hours with short break to administer drugs. Images were acquired at 10-second intervals. A. Treatment with LiCl. B. Treatment with SB216763. C and D. Quantification of the numbers of moving mitochondria during the period of observation (n = 5). Only mitochondria that moved through the entire field of view were calculated. E and F. Quantification of mean velocities of moving mitochondria (n = 5). G. Western blot analysis of acetylated tubulin in extracts from hippocampal neurons that were treated with LiCl or SB216763. H. Quantification of Western blot shown in G. I. Western blot analysis of acetylated tubulin, phosphorylated Akt (pAkt), and phosphorylated GSK3β (pGSK3β) in extracts from hippocampal neurons that were treated with Akt inhibitor. J. Quantification of Western blot shown in I.
Figure 4.
Inhibition of GSK3β decreases protein levels and activity of HDAC6 in hippocampal neurons.
A. Western blot analysis of protein levels of HDAC6 in hippocampal neurons that were treated with LiCl (10 mM) or SB216763 (2 µM) for one hour. B. Quantification of Western blot shown in A (n = 3). C. HDAC6 activity assay of total protein lysate from hippocampal neurons that were treated with LiCl (10 mM) or SB216763 (2 µM) for one hour. D. HDAC6 activity assay of total protein lysate from hippocampal neurons that were treated with Akt inhibitor VIII (5 µM) for one hour. Efficiencies of the separation of cytoplasmic and nuclear phases in C and D are shown by Western blot using anti-GAPDH antibodies.
Figure 5.
HDAC6 and GSK3β are co-localized in hippocampal neurons; HDAC6 serine-22 phosphorylation levels are modulated by GSK3β.
A–F. Immunostaining of HDAC6 and GSK3β in hippocampal neurons. F. Plot of co-localization generated from confocal image data collected from four neurons (six Z levels per cell; total sample size ∼24). G. Immunoprecipitation of GSK3β and HDAC6, respectively. H–J. Confocal images of immunostained acetylated tubulin and phosphorylated HDAC6 (anti-phosphoserine 22) in hippocampal neurons. H. Before treatment. I. After treatment with SB216763. J. After treatment with Akt inhibitor VIII (5 µM). K, L and M. Quantification of images H–J. Mean values of relative pixel intensities are shown.
Figure 6.
5-HT signals increase levels of Kinesin-1B and acetylated tubulin in mitochondrial fractions isolated from hippocampal neurons.
A, C. 5-HT or 8-OH-DPAT increases acetylation of tubulin in hippocampal neurons. B, D. Fluoxetine increases acetylation of tubulin in hippocampal neurons. E, F, G, H. 5-HT, 8-OH-DPAT, or fluoxetine increase the amount of kinesin1-B (top panel) and acetylated tubulin (bottom panel) found in a mitochondrially-enriched fraction. Mitochondrial protein levels were normalized to VDAC. Quantifications of Western blots were based on multiple experiments (n≥3).