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Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration.

Siudeja K, Srinivasan B, Xu L, Rana A, de Jong J, Nollen EA, Jackowski S, Sanford L, Hayflick S, Sibon OC - EMBO Mol Med (2011)

Bottom Line: Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN.Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models.We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Radiation and Stress Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

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Decreased levels of acetylated histones and tubulin are associated with decreased survival and decreased locomotor function of dPANK/Fbl mutant larvaeExtracts of WT and dPANK/Fbl homozygous third instar larvae were analysed for their levels of acetylated proteins using acetyl-Lys antibody. Tubulin was used as a loading control and the dPANK antibody was used to demonstrate the reduced expression of dPANK/Fbl in the mutant larvae. Addition of 0.2 µM TSA to the larval food resulted in increased levels of acetylated histones and tubulin.Quantification of the relative intensity of the 55 kDa band (corresponding to acetyl-tubulin) and <17 kDa bands (corresponding to acetyl-histones) in larvae extracts.Ability of larvae to crawl a certain distance in 9 min was measured as previously described (Rana et al, 2010). Larval crawling assay was performed in WT larvae and in dPANK/Fbl homozygous mutant larvae untreated or fed with the HDAC inhibitor TSA (0.2 µM).dPANK/Fbl/TM3 males and females were crossed and various concentrations of TSA were added to the food. The number of homozygous (dPANK/Fbl/dPANK/Fbl) versus heterozygous dPANK/Fbl/TM3 adults which eclosed was counted.
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fig05: Decreased levels of acetylated histones and tubulin are associated with decreased survival and decreased locomotor function of dPANK/Fbl mutant larvaeExtracts of WT and dPANK/Fbl homozygous third instar larvae were analysed for their levels of acetylated proteins using acetyl-Lys antibody. Tubulin was used as a loading control and the dPANK antibody was used to demonstrate the reduced expression of dPANK/Fbl in the mutant larvae. Addition of 0.2 µM TSA to the larval food resulted in increased levels of acetylated histones and tubulin.Quantification of the relative intensity of the 55 kDa band (corresponding to acetyl-tubulin) and <17 kDa bands (corresponding to acetyl-histones) in larvae extracts.Ability of larvae to crawl a certain distance in 9 min was measured as previously described (Rana et al, 2010). Larval crawling assay was performed in WT larvae and in dPANK/Fbl homozygous mutant larvae untreated or fed with the HDAC inhibitor TSA (0.2 µM).dPANK/Fbl/TM3 males and females were crossed and various concentrations of TSA were added to the food. The number of homozygous (dPANK/Fbl/dPANK/Fbl) versus heterozygous dPANK/Fbl/TM3 adults which eclosed was counted.

Mentions: The Drosophila model for PKAN is further characterized by a decreased survival rate, neurodegeneration and by impaired locomotor function (Afshar et al, 2001; Bosveld et al, 2008; Rana et al, 2010; Wu et al, 2009). The impaired locomotor function is most likely (at least partly) caused by the neurodegeneration. We next investigated whether these phenotypes also correlate with decreased acetylation levels. Especially acetylation of tubulin and histones as we report here for dPANK/Fbl depleted cells have previously been shown by others to be associated with neurodegeneration and with abnormal neuronal functioning (Akella et al, 2010; Creppe et al, 2009; Dompierre et al, 2007; Fischer et al, 2010; Kontopoulos et al, 2006; Monti et al, 2009; Saha and Pahan, 2006; Shida et al, 2010). In the experiments as described above Drosophila S2 cultured cells were used and first we tested whether in Drosophila dPANK/fbl mutant whole organisms (Drosophila PKAN model) levels of acetylated tubulin and histones were also decreased. Western blot analysis using extracts of third instar larvae indeed demonstrated that levels of acetylated tubulin and histones were decreased in dPANK/fbl homozygous larvae as compared to WT larvae (Fig 5A compare lane 1 and lane 3, for quantification see Fig 5B). Homozygous dPANK/fbl flies show a reduced eclosion rate, evidenced by the relative low number of homozygous adults compared to heterozygous adults (the ratio heterozygous:homozygous adult survivors is 16, whereas, based on genetic inheritance this is expected to be 2). First we investigated whether addition of various HDAC inhibitors (valproic acid (VPA), sodium phenylbutyrate (PBA) or TSA to the larval food increased the eclosion rate of homozygous dPANK/fbl flies. VPA and PBA did not result in a significant rescue (Fig S5 of Supporting information) however; TSA addition increased the survival rate of the homozygous mutant progeny in a concentration-dependent manner (Fig 5D). VPA and PBA could only be used in relatively low concentrations, because the concentrations commonly used for an efficient HDAC inhibition (above 1 mM) induced lethality when fed during larval development. TSA, on the other hand, is less toxic. Moreover, TSA is a potent and broad-spectrum inhibitor acting on all Drosophila HDACs (Cho et al, 2005; Foglietti et al, 2006). The most effective concentration of TSA (0.2 µM) was used for further studies and we demonstrated that this induces a partial restoration of the decreased levels of acetylated tubulin and histones in dPANK/fbl mutant larvae (Fig 5A and B). This coincided with an increase in locomotor function assessed by larval crawling as a read-out assay (Fig 5C). These data suggest a correlation between tubulin- and histone-acetylation levels and dPANK/fbl mutant phenotypes. These data are however not conclusive as to whether restoration of only these specific proteins is sufficient for improvement of locomotor function and survival, because acetylation levels of other proteins may also restore upon TSA feeding. Nonetheless, the tight correlation between CoA levels, acetylation of tubulins and histones and the specific phenotypes in dPANK/Fbl depleted cells and flies suggests that an altered status of acetylation of specific proteins may explain the pleiotropic mutant phenotype.


Impaired Coenzyme A metabolism affects histone and tubulin acetylation in Drosophila and human cell models of pantothenate kinase associated neurodegeneration.

Siudeja K, Srinivasan B, Xu L, Rana A, de Jong J, Nollen EA, Jackowski S, Sanford L, Hayflick S, Sibon OC - EMBO Mol Med (2011)

Decreased levels of acetylated histones and tubulin are associated with decreased survival and decreased locomotor function of dPANK/Fbl mutant larvaeExtracts of WT and dPANK/Fbl homozygous third instar larvae were analysed for their levels of acetylated proteins using acetyl-Lys antibody. Tubulin was used as a loading control and the dPANK antibody was used to demonstrate the reduced expression of dPANK/Fbl in the mutant larvae. Addition of 0.2 µM TSA to the larval food resulted in increased levels of acetylated histones and tubulin.Quantification of the relative intensity of the 55 kDa band (corresponding to acetyl-tubulin) and <17 kDa bands (corresponding to acetyl-histones) in larvae extracts.Ability of larvae to crawl a certain distance in 9 min was measured as previously described (Rana et al, 2010). Larval crawling assay was performed in WT larvae and in dPANK/Fbl homozygous mutant larvae untreated or fed with the HDAC inhibitor TSA (0.2 µM).dPANK/Fbl/TM3 males and females were crossed and various concentrations of TSA were added to the food. The number of homozygous (dPANK/Fbl/dPANK/Fbl) versus heterozygous dPANK/Fbl/TM3 adults which eclosed was counted.
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fig05: Decreased levels of acetylated histones and tubulin are associated with decreased survival and decreased locomotor function of dPANK/Fbl mutant larvaeExtracts of WT and dPANK/Fbl homozygous third instar larvae were analysed for their levels of acetylated proteins using acetyl-Lys antibody. Tubulin was used as a loading control and the dPANK antibody was used to demonstrate the reduced expression of dPANK/Fbl in the mutant larvae. Addition of 0.2 µM TSA to the larval food resulted in increased levels of acetylated histones and tubulin.Quantification of the relative intensity of the 55 kDa band (corresponding to acetyl-tubulin) and <17 kDa bands (corresponding to acetyl-histones) in larvae extracts.Ability of larvae to crawl a certain distance in 9 min was measured as previously described (Rana et al, 2010). Larval crawling assay was performed in WT larvae and in dPANK/Fbl homozygous mutant larvae untreated or fed with the HDAC inhibitor TSA (0.2 µM).dPANK/Fbl/TM3 males and females were crossed and various concentrations of TSA were added to the food. The number of homozygous (dPANK/Fbl/dPANK/Fbl) versus heterozygous dPANK/Fbl/TM3 adults which eclosed was counted.
Mentions: The Drosophila model for PKAN is further characterized by a decreased survival rate, neurodegeneration and by impaired locomotor function (Afshar et al, 2001; Bosveld et al, 2008; Rana et al, 2010; Wu et al, 2009). The impaired locomotor function is most likely (at least partly) caused by the neurodegeneration. We next investigated whether these phenotypes also correlate with decreased acetylation levels. Especially acetylation of tubulin and histones as we report here for dPANK/Fbl depleted cells have previously been shown by others to be associated with neurodegeneration and with abnormal neuronal functioning (Akella et al, 2010; Creppe et al, 2009; Dompierre et al, 2007; Fischer et al, 2010; Kontopoulos et al, 2006; Monti et al, 2009; Saha and Pahan, 2006; Shida et al, 2010). In the experiments as described above Drosophila S2 cultured cells were used and first we tested whether in Drosophila dPANK/fbl mutant whole organisms (Drosophila PKAN model) levels of acetylated tubulin and histones were also decreased. Western blot analysis using extracts of third instar larvae indeed demonstrated that levels of acetylated tubulin and histones were decreased in dPANK/fbl homozygous larvae as compared to WT larvae (Fig 5A compare lane 1 and lane 3, for quantification see Fig 5B). Homozygous dPANK/fbl flies show a reduced eclosion rate, evidenced by the relative low number of homozygous adults compared to heterozygous adults (the ratio heterozygous:homozygous adult survivors is 16, whereas, based on genetic inheritance this is expected to be 2). First we investigated whether addition of various HDAC inhibitors (valproic acid (VPA), sodium phenylbutyrate (PBA) or TSA to the larval food increased the eclosion rate of homozygous dPANK/fbl flies. VPA and PBA did not result in a significant rescue (Fig S5 of Supporting information) however; TSA addition increased the survival rate of the homozygous mutant progeny in a concentration-dependent manner (Fig 5D). VPA and PBA could only be used in relatively low concentrations, because the concentrations commonly used for an efficient HDAC inhibition (above 1 mM) induced lethality when fed during larval development. TSA, on the other hand, is less toxic. Moreover, TSA is a potent and broad-spectrum inhibitor acting on all Drosophila HDACs (Cho et al, 2005; Foglietti et al, 2006). The most effective concentration of TSA (0.2 µM) was used for further studies and we demonstrated that this induces a partial restoration of the decreased levels of acetylated tubulin and histones in dPANK/fbl mutant larvae (Fig 5A and B). This coincided with an increase in locomotor function assessed by larval crawling as a read-out assay (Fig 5C). These data suggest a correlation between tubulin- and histone-acetylation levels and dPANK/fbl mutant phenotypes. These data are however not conclusive as to whether restoration of only these specific proteins is sufficient for improvement of locomotor function and survival, because acetylation levels of other proteins may also restore upon TSA feeding. Nonetheless, the tight correlation between CoA levels, acetylation of tubulins and histones and the specific phenotypes in dPANK/Fbl depleted cells and flies suggests that an altered status of acetylation of specific proteins may explain the pleiotropic mutant phenotype.

Bottom Line: Previously, we observed reduced Coenzyme A levels in a Drosophila model for PKAN.Here we investigate whether decreased levels of the central metabolite Coenzyme A induce alterations in protein acetylation and whether this correlates with specific phenotypes of PKAN models.We show that in various organisms proper Coenzyme A metabolism is required for maintenance of histone- and tubulin acetylation, and decreased acetylation of these proteins is associated with an impaired DNA damage response, decreased locomotor function and decreased survival.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, Radiation and Stress Cell Biology, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands.

Show MeSH
Related in: MedlinePlus