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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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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.
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pone-0010848-g002: 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.

Mentions: In agreement with an earlier report using non-neuronal cells [14], Western blot analysis of acetylated tubulin in extracts from hippocampal neurons showed that treatment with tubacin (20 µM) increased levels of acetylated tubulin (Fig. 2A, lane 2). Treatment with TSA (10 µM) had a similar effect (Fig. 2A, lane3) and, as expected, the administration of niltubacin (20 µM) did not change levels of tubulin acetylation, relative to controls (Fig. 2B, lane 2). Total levels of kinesin-1B and tubulin were unaffected (Fig. 2A and B).


HDAC6 regulates mitochondrial transport in hippocampal neurons.

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

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2877100&req=5

pone-0010848-g002: 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.
Mentions: In agreement with an earlier report using non-neuronal cells [14], Western blot analysis of acetylated tubulin in extracts from hippocampal neurons showed that treatment with tubacin (20 µM) increased levels of acetylated tubulin (Fig. 2A, lane 2). Treatment with TSA (10 µM) had a similar effect (Fig. 2A, lane3) and, as expected, the administration of niltubacin (20 µM) did not change levels of tubulin acetylation, relative to controls (Fig. 2B, lane 2). Total levels of kinesin-1B and tubulin were unaffected (Fig. 2A and B).

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