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Genetic knock-down of HDAC3 does not modify disease-related phenotypes in a mouse model of Huntington's disease.

Moumné L, Campbell K, Howland D, Ouyang Y, Bates GP - PLoS ONE (2012)

Bottom Line: However potent pan-HDAC inhibitors such as SAHA display toxic side-effects.We found that Hdac3 knock-down does not ameliorate physiological or behavioural phenotypes and has no effect on molecular changes including dysregulated transcripts.We conclude that HDAC3 should not be considered as the major mediator of the beneficial effect induced by SAHA and other HDAC inhibitors in HD.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical and Molecular Genetics, King's College London, London, United Kingdom.

ABSTRACT
Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by an expansion of a CAG/polyglutamine repeat for which there are no disease modifying treatments. In recent years, transcriptional dysregulation has emerged as a pathogenic process that appears early in disease progression and has been recapitulated across multiple HD models. Altered histone acetylation has been proposed to underlie this transcriptional dysregulation and histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA), have been shown to improve polyglutamine-dependent phenotypes in numerous HD models. However potent pan-HDAC inhibitors such as SAHA display toxic side-effects. To better understand the mechanism underlying this potential therapeutic benefit and to dissociate the beneficial and toxic effects of SAHA, we set out to identify the specific HDAC(s) involved in this process. For this purpose, we are exploring the effect of the genetic reduction of specific HDACs on HD-related phenotypes in the R6/2 mouse model of HD. The study presented here focuses on HDAC3, which, as a class I HDAC, is one of the preferred targets of SAHA and is directly involved in histone deacetylation. To evaluate a potential benefit of Hdac3 genetic reduction in R6/2, we generated a mouse carrying a critical deletion in the Hdac3 gene. We confirmed that the complete knock-out of Hdac3 is embryonic lethal. To test the effects of HDAC3 inhibition, we used Hdac3(+/-) heterozygotes to reduce nuclear HDAC3 levels in R6/2 mice. We found that Hdac3 knock-down does not ameliorate physiological or behavioural phenotypes and has no effect on molecular changes including dysregulated transcripts. We conclude that HDAC3 should not be considered as the major mediator of the beneficial effect induced by SAHA and other HDAC inhibitors in HD.

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Hdac3 mRNA and protein expression in Hdac3+/− heterozygous mouse brain.(A) Hdac3 mRNA expression levels in 15 week old mouse cortex, cerebellum and striatum are shown as a relative expression ratio to the WT level. Hdac3+/− (red) and Dbl (purple) mice express the mRNA at 50% of the WT (blue) and R6/2 (green) levels in all brain regions. Error bars correspond to S.E.M. (n = 8) ***p<0.001. (B) Western blot showing the expression of HDAC3 protein in the cytoplasmic (C) and the nuclear (N) fraction of WT mouse whole brain. Antibodies to α-tubulin (cytoplasmic) and histone H4 (nuclear) were used to control for the purity of the fractions. (C) Representative western blot and (D) quantification of cytoplasmic and nuclear fraction prepared from WT (blue), Hdac3+/− (red), R6/2 (green) and Dbl (purple) 15 week old-mouse whole brains. Antibodies to α-tubulin (cytoplasmic) and histone H4 (nuclear) were used as both purity and loading controls. Cytoplasmic HDAC3 was not affected by Hdac3 deletion whereas nuclear HDAC3 was reduced to 60% of the WT level. Error bars correspond S.E.M. (n = 3) *p<0.05.
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pone-0031080-g002: Hdac3 mRNA and protein expression in Hdac3+/− heterozygous mouse brain.(A) Hdac3 mRNA expression levels in 15 week old mouse cortex, cerebellum and striatum are shown as a relative expression ratio to the WT level. Hdac3+/− (red) and Dbl (purple) mice express the mRNA at 50% of the WT (blue) and R6/2 (green) levels in all brain regions. Error bars correspond to S.E.M. (n = 8) ***p<0.001. (B) Western blot showing the expression of HDAC3 protein in the cytoplasmic (C) and the nuclear (N) fraction of WT mouse whole brain. Antibodies to α-tubulin (cytoplasmic) and histone H4 (nuclear) were used to control for the purity of the fractions. (C) Representative western blot and (D) quantification of cytoplasmic and nuclear fraction prepared from WT (blue), Hdac3+/− (red), R6/2 (green) and Dbl (purple) 15 week old-mouse whole brains. Antibodies to α-tubulin (cytoplasmic) and histone H4 (nuclear) were used as both purity and loading controls. Cytoplasmic HDAC3 was not affected by Hdac3 deletion whereas nuclear HDAC3 was reduced to 60% of the WT level. Error bars correspond S.E.M. (n = 3) *p<0.05.

Mentions: It was important first to confirm that the Hdac3 expression level was reduced in Hdac3+/− mice as it has been previously shown that Hdac1, another class I HDAC, autoregulates its expression to wild-type (WT) levels in Hdac1+/− heterozygous mice [37]. It was also important to determine whether Hdac3 levels might be regulated by the R6/2 transprotein. Therefore, we crossed R6/2 males to Hdac3+/− heterozygous females to generate WT, Hdac3+/−, R6/2 and R6/2::Hdac3+/− double mutant (Dbl) mice and performed RT-qPCR on cDNA prepared from cortex, cerebellum and striatum of 15-week old mice. The RT-qPCR primer sequences were located within the deletion (at the junction between exon 14 and 15) so that only the WT allele can be detected. We observed a significant reduction of Hdac3 mRNA to 50% of the WT level in all the brain regions irrespective of the presence of the R6/2 transprotein (Fig. 2A). In order to verify the specificity of Hdac3 reduction we analyzed the expression level of the other Hdacs by RT-qPCR and showed that these were not changed in Hdac3+/− mice compared to WT (Fig. S1).


Genetic knock-down of HDAC3 does not modify disease-related phenotypes in a mouse model of Huntington's disease.

Moumné L, Campbell K, Howland D, Ouyang Y, Bates GP - PLoS ONE (2012)

Hdac3 mRNA and protein expression in Hdac3+/− heterozygous mouse brain.(A) Hdac3 mRNA expression levels in 15 week old mouse cortex, cerebellum and striatum are shown as a relative expression ratio to the WT level. Hdac3+/− (red) and Dbl (purple) mice express the mRNA at 50% of the WT (blue) and R6/2 (green) levels in all brain regions. Error bars correspond to S.E.M. (n = 8) ***p<0.001. (B) Western blot showing the expression of HDAC3 protein in the cytoplasmic (C) and the nuclear (N) fraction of WT mouse whole brain. Antibodies to α-tubulin (cytoplasmic) and histone H4 (nuclear) were used to control for the purity of the fractions. (C) Representative western blot and (D) quantification of cytoplasmic and nuclear fraction prepared from WT (blue), Hdac3+/− (red), R6/2 (green) and Dbl (purple) 15 week old-mouse whole brains. Antibodies to α-tubulin (cytoplasmic) and histone H4 (nuclear) were used as both purity and loading controls. Cytoplasmic HDAC3 was not affected by Hdac3 deletion whereas nuclear HDAC3 was reduced to 60% of the WT level. Error bars correspond S.E.M. (n = 3) *p<0.05.
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Related In: Results  -  Collection

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pone-0031080-g002: Hdac3 mRNA and protein expression in Hdac3+/− heterozygous mouse brain.(A) Hdac3 mRNA expression levels in 15 week old mouse cortex, cerebellum and striatum are shown as a relative expression ratio to the WT level. Hdac3+/− (red) and Dbl (purple) mice express the mRNA at 50% of the WT (blue) and R6/2 (green) levels in all brain regions. Error bars correspond to S.E.M. (n = 8) ***p<0.001. (B) Western blot showing the expression of HDAC3 protein in the cytoplasmic (C) and the nuclear (N) fraction of WT mouse whole brain. Antibodies to α-tubulin (cytoplasmic) and histone H4 (nuclear) were used to control for the purity of the fractions. (C) Representative western blot and (D) quantification of cytoplasmic and nuclear fraction prepared from WT (blue), Hdac3+/− (red), R6/2 (green) and Dbl (purple) 15 week old-mouse whole brains. Antibodies to α-tubulin (cytoplasmic) and histone H4 (nuclear) were used as both purity and loading controls. Cytoplasmic HDAC3 was not affected by Hdac3 deletion whereas nuclear HDAC3 was reduced to 60% of the WT level. Error bars correspond S.E.M. (n = 3) *p<0.05.
Mentions: It was important first to confirm that the Hdac3 expression level was reduced in Hdac3+/− mice as it has been previously shown that Hdac1, another class I HDAC, autoregulates its expression to wild-type (WT) levels in Hdac1+/− heterozygous mice [37]. It was also important to determine whether Hdac3 levels might be regulated by the R6/2 transprotein. Therefore, we crossed R6/2 males to Hdac3+/− heterozygous females to generate WT, Hdac3+/−, R6/2 and R6/2::Hdac3+/− double mutant (Dbl) mice and performed RT-qPCR on cDNA prepared from cortex, cerebellum and striatum of 15-week old mice. The RT-qPCR primer sequences were located within the deletion (at the junction between exon 14 and 15) so that only the WT allele can be detected. We observed a significant reduction of Hdac3 mRNA to 50% of the WT level in all the brain regions irrespective of the presence of the R6/2 transprotein (Fig. 2A). In order to verify the specificity of Hdac3 reduction we analyzed the expression level of the other Hdacs by RT-qPCR and showed that these were not changed in Hdac3+/− mice compared to WT (Fig. S1).

Bottom Line: However potent pan-HDAC inhibitors such as SAHA display toxic side-effects.We found that Hdac3 knock-down does not ameliorate physiological or behavioural phenotypes and has no effect on molecular changes including dysregulated transcripts.We conclude that HDAC3 should not be considered as the major mediator of the beneficial effect induced by SAHA and other HDAC inhibitors in HD.

View Article: PubMed Central - PubMed

Affiliation: Department of Medical and Molecular Genetics, King's College London, London, United Kingdom.

ABSTRACT
Huntington's disease (HD) is an autosomal dominant progressive neurodegenerative disorder caused by an expansion of a CAG/polyglutamine repeat for which there are no disease modifying treatments. In recent years, transcriptional dysregulation has emerged as a pathogenic process that appears early in disease progression and has been recapitulated across multiple HD models. Altered histone acetylation has been proposed to underlie this transcriptional dysregulation and histone deacetylase (HDAC) inhibitors, such as suberoylanilide hydroxamic acid (SAHA), have been shown to improve polyglutamine-dependent phenotypes in numerous HD models. However potent pan-HDAC inhibitors such as SAHA display toxic side-effects. To better understand the mechanism underlying this potential therapeutic benefit and to dissociate the beneficial and toxic effects of SAHA, we set out to identify the specific HDAC(s) involved in this process. For this purpose, we are exploring the effect of the genetic reduction of specific HDACs on HD-related phenotypes in the R6/2 mouse model of HD. The study presented here focuses on HDAC3, which, as a class I HDAC, is one of the preferred targets of SAHA and is directly involved in histone deacetylation. To evaluate a potential benefit of Hdac3 genetic reduction in R6/2, we generated a mouse carrying a critical deletion in the Hdac3 gene. We confirmed that the complete knock-out of Hdac3 is embryonic lethal. To test the effects of HDAC3 inhibition, we used Hdac3(+/-) heterozygotes to reduce nuclear HDAC3 levels in R6/2 mice. We found that Hdac3 knock-down does not ameliorate physiological or behavioural phenotypes and has no effect on molecular changes including dysregulated transcripts. We conclude that HDAC3 should not be considered as the major mediator of the beneficial effect induced by SAHA and other HDAC inhibitors in HD.

Show MeSH
Related in: MedlinePlus