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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 genetic reduction does not reduce HTT aggregation in R6/2 mouse brain.(A) The SEPRION ligand based ELISA assay was used to quantify HTT aggregation in the cortex, hippocampus and brain stem of 4, 9 and 15 week-old mice. The graphs represent the microtitre-plate reading of R6/2 (green) and Dbl (purple) lysates. Background readings obtained with WT and Hdac3 lysates were comparable to water. Aggregation levels augment with age but are not modified by Hdac3 reduction. Error bars correspond to S.E.M. (n>6). (B) Representative western blot of hippocampal lysates at 4, 9 and 15 weeks of age. The aggregated HTT fraction (stacking gel) augments with age whereas the soluble fraction decreases with age. α-tubulin was used as a loading control. (C) Quantification of (B). Soluble HTT is represented as a percentage of the soluble fraction in R6/2. Error bars correspond to S.E.M. (n = 6). The same color code (R6/2 = green; Dbl = purple) is used in (A) and (C).
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pone-0031080-g005: Hdac3 genetic reduction does not reduce HTT aggregation in R6/2 mouse brain.(A) The SEPRION ligand based ELISA assay was used to quantify HTT aggregation in the cortex, hippocampus and brain stem of 4, 9 and 15 week-old mice. The graphs represent the microtitre-plate reading of R6/2 (green) and Dbl (purple) lysates. Background readings obtained with WT and Hdac3 lysates were comparable to water. Aggregation levels augment with age but are not modified by Hdac3 reduction. Error bars correspond to S.E.M. (n>6). (B) Representative western blot of hippocampal lysates at 4, 9 and 15 weeks of age. The aggregated HTT fraction (stacking gel) augments with age whereas the soluble fraction decreases with age. α-tubulin was used as a loading control. (C) Quantification of (B). Soluble HTT is represented as a percentage of the soluble fraction in R6/2. Error bars correspond to S.E.M. (n = 6). The same color code (R6/2 = green; Dbl = purple) is used in (A) and (C).

Mentions: HTT aggregation is a hallmark of brain pathology in HD and has been consistently observed in all HD models including the R6/2 mouse. Aggregation can be detected in several R6/2 brain regions from 3–4 weeks of age and increases with age [11]. In order to evaluate whether Hdac3 reduction has an effect on this aggregation, we used a SEPRION ligand-based ELISA that provides a highly quantitative assay for measuring HTT aggregation in mouse brains [14]. Aggregates were captured from proteins extracted from the cortex, hippocampus and brain stem of 4, 9 and 15 week-old mice and detected with the MW8 antibody [39]. As expected, HTT aggregation was detectable at 4 weeks in all three brain regions, increased with age and this was not modified by Hdac3 reduction. Levels of soluble HTT in these lysates can be quantified by western blot using the HTT specific antibody S830 [40]. The soluble trans-protein migrates at around 95 kDa whereas the aggregated protein remains in the stacking gel. Fig. 5B shows the results that we obtained for hippocampi from mice of 4, 9 and 15 weeks of age. The soluble fraction decreased with age in both R6/2 and Dbl mice and did not differ significantly between these genotypes at any time point (Fig. 5C). Similarly, a qualitative difference of the aggregated fraction was not detected. Taken together, the data obtained from the ELISA and western blotting revealed that Hdac3 genetic reduction has no effect on HTT aggregation.


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 genetic reduction does not reduce HTT aggregation in R6/2 mouse brain.(A) The SEPRION ligand based ELISA assay was used to quantify HTT aggregation in the cortex, hippocampus and brain stem of 4, 9 and 15 week-old mice. The graphs represent the microtitre-plate reading of R6/2 (green) and Dbl (purple) lysates. Background readings obtained with WT and Hdac3 lysates were comparable to water. Aggregation levels augment with age but are not modified by Hdac3 reduction. Error bars correspond to S.E.M. (n>6). (B) Representative western blot of hippocampal lysates at 4, 9 and 15 weeks of age. The aggregated HTT fraction (stacking gel) augments with age whereas the soluble fraction decreases with age. α-tubulin was used as a loading control. (C) Quantification of (B). Soluble HTT is represented as a percentage of the soluble fraction in R6/2. Error bars correspond to S.E.M. (n = 6). The same color code (R6/2 = green; Dbl = purple) is used in (A) and (C).
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Related In: Results  -  Collection

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pone-0031080-g005: Hdac3 genetic reduction does not reduce HTT aggregation in R6/2 mouse brain.(A) The SEPRION ligand based ELISA assay was used to quantify HTT aggregation in the cortex, hippocampus and brain stem of 4, 9 and 15 week-old mice. The graphs represent the microtitre-plate reading of R6/2 (green) and Dbl (purple) lysates. Background readings obtained with WT and Hdac3 lysates were comparable to water. Aggregation levels augment with age but are not modified by Hdac3 reduction. Error bars correspond to S.E.M. (n>6). (B) Representative western blot of hippocampal lysates at 4, 9 and 15 weeks of age. The aggregated HTT fraction (stacking gel) augments with age whereas the soluble fraction decreases with age. α-tubulin was used as a loading control. (C) Quantification of (B). Soluble HTT is represented as a percentage of the soluble fraction in R6/2. Error bars correspond to S.E.M. (n = 6). The same color code (R6/2 = green; Dbl = purple) is used in (A) and (C).
Mentions: HTT aggregation is a hallmark of brain pathology in HD and has been consistently observed in all HD models including the R6/2 mouse. Aggregation can be detected in several R6/2 brain regions from 3–4 weeks of age and increases with age [11]. In order to evaluate whether Hdac3 reduction has an effect on this aggregation, we used a SEPRION ligand-based ELISA that provides a highly quantitative assay for measuring HTT aggregation in mouse brains [14]. Aggregates were captured from proteins extracted from the cortex, hippocampus and brain stem of 4, 9 and 15 week-old mice and detected with the MW8 antibody [39]. As expected, HTT aggregation was detectable at 4 weeks in all three brain regions, increased with age and this was not modified by Hdac3 reduction. Levels of soluble HTT in these lysates can be quantified by western blot using the HTT specific antibody S830 [40]. The soluble trans-protein migrates at around 95 kDa whereas the aggregated protein remains in the stacking gel. Fig. 5B shows the results that we obtained for hippocampi from mice of 4, 9 and 15 weeks of age. The soluble fraction decreased with age in both R6/2 and Dbl mice and did not differ significantly between these genotypes at any time point (Fig. 5C). Similarly, a qualitative difference of the aggregated fraction was not detected. Taken together, the data obtained from the ELISA and western blotting revealed that Hdac3 genetic reduction has no effect on HTT aggregation.

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