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HDAC6 regulates mitochondrial transport in hippocampal neurons.

Chen S, Owens GC, Makarenkova H, Edelman DB - PLoS ONE (2010)

Bottom Line: We found that the presence of tubacin, a specific HDAC6 inhibitor, dramatically enhanced mitochondrial movement in hippocampal neurons, whereas niltubacin, an inactive tubacin analog, had no effect.Compared to control cultures, higher levels of acetylated tubulin were found in neurons treated with tubacin, and more kinesin-1 was associated with mitochondria isolated from these neurons.GSK3beta was found to co-localize with HDAC6 in hippocampal neurons, and inhibition of GSK3beta resulted in decreased binding of antibody to phosphoserine-22, a potential GSK3beta phosphorylation site in HDAC6.

View Article: PubMed Central - PubMed

Affiliation: The Neurosciences Institute, San Diego, California, USA. schen@nsi.edu

ABSTRACT

Background: Tubulin is a major substrate of the cytoplasmic class II histone deacetylase HDAC6. Inhibition of HDAC6 results in higher levels of acetylated tubulin and enhanced binding of the motor protein kinesin-1 to tubulin, which promotes transport of cargoes along microtubules. Microtubule-dependent intracellular trafficking may therefore be regulated by modulating the activity of HDAC6. We have shown previously that the neuromodulator serotonin increases mitochondrial movement in hippocampal neurons via the Akt-GSK3beta signaling pathway. Here, we demonstrate a role for HDAC6 in this signaling pathway.

Methodology/principal findings: We found that the presence of tubacin, a specific HDAC6 inhibitor, dramatically enhanced mitochondrial movement in hippocampal neurons, whereas niltubacin, an inactive tubacin analog, had no effect. Compared to control cultures, higher levels of acetylated tubulin were found in neurons treated with tubacin, and more kinesin-1 was associated with mitochondria isolated from these neurons. Inhibition of GSK3beta decreased cytoplasmic deacetylase activity and increased tubulin acetylation, whereas blockade of Akt, which phosphorylates and down-regulates GSK3beta, increased cytoplasmic deacetylase activity and decreased tubulin acetylation. Concordantly, the administration of 5-HT, 8-OH-DPAT (a specific 5-HT1A receptor agonist), or fluoxetine (a 5-HT reuptake inhibitor) increased tubulin acetylation. GSK3beta was found to co-localize with HDAC6 in hippocampal neurons, and inhibition of GSK3beta resulted in decreased binding of antibody to phosphoserine-22, a potential GSK3beta phosphorylation site in HDAC6. GSK3beta may therefore regulate HDAC6 activity by phosphorylation.

Conclusions/significance: This study demonstrates that HDAC6 plays an important role in the modulation of mitochondrial transport. The link between HDAC6 and GSK3beta, established here, has important implications for our understanding of neurodegenerative disorders. In particular, abnormal mitochondrial transport, which has been observed in such disorders as Alzheimer's disease and Parkinson's disease, could result from the misregulation of HDAC6 by GSK3beta. HDAC6 may therefore constitute an attractive target in the treatment of these disorders.

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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.
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pone-0010848-g003: 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.

Mentions: In a previous study, we found that inhibition of GSK3β dramatically stimulated mitochondrial movement [12]. The fact that many substrates of GSK3β are cytoskeleton-related proteins [16] prompted us to investigate the effects of GSK3β inhibition on the acetylation of tubulin. We found that inhibiting GSK3β with lithium chloride (LiCl, 10 mM) resulted in both an increase in the level of acetylated tubulin and the amount of kinesin-1 associated with mitochondria (Fig. 2B, lane 3; Fig. 2F, lane 3). These results closely resemble the effects of inhibiting HDAC6 using tubacin or TSA (Fig. 2A, lanes 2 and 3; Fig. 2E, lanes 2 and 3). Using two different GSK3β inhibitors, we confirmed that blocking activity greatly enhanced mitochondrial movement, as shown by the kymographs presented in Fig. 3A and B (Movies S10, S11, S12, S13, S14, S15). Quantification of the number of moving mitochondria and average velocity are shown in Fig. 3C–F. In parallel cultures, inhibition of GSK3β led to an approximately 60% increase in the acetylation of tubulin (Fig. 3G and H). In contrast, levels of acetylated tubulin declined by approximately 40% when GSK3β activity was increased by inhibiting Akt activity (Fig. 3I and J). These results are consistent with the idea that the Akt-GSK3β signaling pathway may control mitochondrial movement in neurons by modulating acetylation of microtubules via the regulation of HDAC6.


HDAC6 regulates mitochondrial transport in hippocampal neurons.

Chen S, Owens GC, Makarenkova H, Edelman DB - PLoS ONE (2010)

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.
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pone-0010848-g003: 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.
Mentions: In a previous study, we found that inhibition of GSK3β dramatically stimulated mitochondrial movement [12]. The fact that many substrates of GSK3β are cytoskeleton-related proteins [16] prompted us to investigate the effects of GSK3β inhibition on the acetylation of tubulin. We found that inhibiting GSK3β with lithium chloride (LiCl, 10 mM) resulted in both an increase in the level of acetylated tubulin and the amount of kinesin-1 associated with mitochondria (Fig. 2B, lane 3; Fig. 2F, lane 3). These results closely resemble the effects of inhibiting HDAC6 using tubacin or TSA (Fig. 2A, lanes 2 and 3; Fig. 2E, lanes 2 and 3). Using two different GSK3β inhibitors, we confirmed that blocking activity greatly enhanced mitochondrial movement, as shown by the kymographs presented in Fig. 3A and B (Movies S10, S11, S12, S13, S14, S15). Quantification of the number of moving mitochondria and average velocity are shown in Fig. 3C–F. In parallel cultures, inhibition of GSK3β led to an approximately 60% increase in the acetylation of tubulin (Fig. 3G and H). In contrast, levels of acetylated tubulin declined by approximately 40% when GSK3β activity was increased by inhibiting Akt activity (Fig. 3I and J). These results are consistent with the idea that the Akt-GSK3β signaling pathway may control mitochondrial movement in neurons by modulating acetylation of microtubules via the regulation of HDAC6.

Bottom Line: We found that the presence of tubacin, a specific HDAC6 inhibitor, dramatically enhanced mitochondrial movement in hippocampal neurons, whereas niltubacin, an inactive tubacin analog, had no effect.Compared to control cultures, higher levels of acetylated tubulin were found in neurons treated with tubacin, and more kinesin-1 was associated with mitochondria isolated from these neurons.GSK3beta was found to co-localize with HDAC6 in hippocampal neurons, and inhibition of GSK3beta resulted in decreased binding of antibody to phosphoserine-22, a potential GSK3beta phosphorylation site in HDAC6.

View Article: PubMed Central - PubMed

Affiliation: The Neurosciences Institute, San Diego, California, USA. schen@nsi.edu

ABSTRACT

Background: Tubulin is a major substrate of the cytoplasmic class II histone deacetylase HDAC6. Inhibition of HDAC6 results in higher levels of acetylated tubulin and enhanced binding of the motor protein kinesin-1 to tubulin, which promotes transport of cargoes along microtubules. Microtubule-dependent intracellular trafficking may therefore be regulated by modulating the activity of HDAC6. We have shown previously that the neuromodulator serotonin increases mitochondrial movement in hippocampal neurons via the Akt-GSK3beta signaling pathway. Here, we demonstrate a role for HDAC6 in this signaling pathway.

Methodology/principal findings: We found that the presence of tubacin, a specific HDAC6 inhibitor, dramatically enhanced mitochondrial movement in hippocampal neurons, whereas niltubacin, an inactive tubacin analog, had no effect. Compared to control cultures, higher levels of acetylated tubulin were found in neurons treated with tubacin, and more kinesin-1 was associated with mitochondria isolated from these neurons. Inhibition of GSK3beta decreased cytoplasmic deacetylase activity and increased tubulin acetylation, whereas blockade of Akt, which phosphorylates and down-regulates GSK3beta, increased cytoplasmic deacetylase activity and decreased tubulin acetylation. Concordantly, the administration of 5-HT, 8-OH-DPAT (a specific 5-HT1A receptor agonist), or fluoxetine (a 5-HT reuptake inhibitor) increased tubulin acetylation. GSK3beta was found to co-localize with HDAC6 in hippocampal neurons, and inhibition of GSK3beta resulted in decreased binding of antibody to phosphoserine-22, a potential GSK3beta phosphorylation site in HDAC6. GSK3beta may therefore regulate HDAC6 activity by phosphorylation.

Conclusions/significance: This study demonstrates that HDAC6 plays an important role in the modulation of mitochondrial transport. The link between HDAC6 and GSK3beta, established here, has important implications for our understanding of neurodegenerative disorders. In particular, abnormal mitochondrial transport, which has been observed in such disorders as Alzheimer's disease and Parkinson's disease, could result from the misregulation of HDAC6 by GSK3beta. HDAC6 may therefore constitute an attractive target in the treatment of these disorders.

Show MeSH
Related in: MedlinePlus