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CpG demethylation enhances alpha-synuclein expression and affects the pathogenesis of Parkinson's disease.

Matsumoto L, Takuma H, Tamaoka A, Kurisaki H, Date H, Tsuji S, Iwata A - PLoS ONE (2010)

Bottom Line: Gene multiplication can cause inherited PD, and promoter polymorphisms that increase SNCA expression are associated with sporadic PD.By using cultured cells, we identified a region of the SNCA CpG island in which the methylation status altered along with increased SNCA expression.This CpG region may function as an intronic regulatory element for SNCA gene.

View Article: PubMed Central - PubMed

Affiliation: Division of Neuroscience, Department of Neurology, Graduate School of Medicine, The University of Tokyo, Bunkyo, Tokyo, Japan.

ABSTRACT

Background: Alpha-synuclein (SNCA) gene expression is an important factor in the pathogenesis of Parkinson's disease (PD). Gene multiplication can cause inherited PD, and promoter polymorphisms that increase SNCA expression are associated with sporadic PD. CpG methylation in the promoter region may also influence SNCA expression.

Methodology/principal findings: By using cultured cells, we identified a region of the SNCA CpG island in which the methylation status altered along with increased SNCA expression. Postmortem brain analysis revealed regional non-specific methylation differences in this CpG region in the anterior cingulate and putamen among controls and PD; however, in the substantia nigra of PD, methylation was significantly decreased.

Conclusions/significance: This CpG region may function as an intronic regulatory element for SNCA gene. Our findings suggest that a novel epigenetic regulatory mechanism controlling SNCA expression influences PD pathogenesis.

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Related in: MedlinePlus

Identification of SNCA CpG islands.A: 293 cells express endogenous alpha-synuclein Immunoblot of cell lysates from SH-SY5Y, 293, and HeLa cells with or without over-expression of wild-type alpha-synuclein (transfection − and +). Arrow indicates alpha-synuclein. B: Quantification of alpha-synuclein expression in the presence of various stimuli. DMSO (−), IL-1β (100 ng/mL), lipopolysaccharides (L2654 and L6529 1 µg/mL), βFGF (100 ng/mL), NGF (100 ng/mL) and dopamine (DA 50 µM) were added to 293 cells for 48 h and relative expression of alpha-synuclein was compared by quantitative RT-PCR. Relative amounts of alpha-synuclein mRNA were normalized to DMSO = 1.0, * P<0.01, ** P<0.001. C: Structure of the SNCA CpG island Entrez gene NC_000004.11 ID was analyzed by CpG island search software (http://www.uscnorris.com/cpgislands2/cpg.aspx.). Non-coding exon1 and coding exon2 are indicated by open arrows. The translation start ATG codon (methionine) located at exon2 is indicated by a closed circle. Two separate CpG islands are indicated by closed bars. D: Methylated-CpG island recovery assay of dopamine treated cells. 293 cells treated with or without dopamine were lysed and methylated. CpGs were enriched by MBD2 protein. Methylated CpG-1 or CpG-2 was detected by PCR before and after enrichment.
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pone-0015522-g001: Identification of SNCA CpG islands.A: 293 cells express endogenous alpha-synuclein Immunoblot of cell lysates from SH-SY5Y, 293, and HeLa cells with or without over-expression of wild-type alpha-synuclein (transfection − and +). Arrow indicates alpha-synuclein. B: Quantification of alpha-synuclein expression in the presence of various stimuli. DMSO (−), IL-1β (100 ng/mL), lipopolysaccharides (L2654 and L6529 1 µg/mL), βFGF (100 ng/mL), NGF (100 ng/mL) and dopamine (DA 50 µM) were added to 293 cells for 48 h and relative expression of alpha-synuclein was compared by quantitative RT-PCR. Relative amounts of alpha-synuclein mRNA were normalized to DMSO = 1.0, * P<0.01, ** P<0.001. C: Structure of the SNCA CpG island Entrez gene NC_000004.11 ID was analyzed by CpG island search software (http://www.uscnorris.com/cpgislands2/cpg.aspx.). Non-coding exon1 and coding exon2 are indicated by open arrows. The translation start ATG codon (methionine) located at exon2 is indicated by a closed circle. Two separate CpG islands are indicated by closed bars. D: Methylated-CpG island recovery assay of dopamine treated cells. 293 cells treated with or without dopamine were lysed and methylated. CpGs were enriched by MBD2 protein. Methylated CpG-1 or CpG-2 was detected by PCR before and after enrichment.

Mentions: Using 293 cells, we tested various drugs that have been reported to alter alpha-synuclein expression. Of 6 reagents tested, 1 lipopolysaccharide and dopamine caused a significant increase in the production of alpha-synuclein (Figure 1B). Because dopamine seemed to enhance alpha-synuclein expression more than the lipopolysaccharide, we chose to analyze its effect on SNCA CpG island methylation.


CpG demethylation enhances alpha-synuclein expression and affects the pathogenesis of Parkinson's disease.

Matsumoto L, Takuma H, Tamaoka A, Kurisaki H, Date H, Tsuji S, Iwata A - PLoS ONE (2010)

Identification of SNCA CpG islands.A: 293 cells express endogenous alpha-synuclein Immunoblot of cell lysates from SH-SY5Y, 293, and HeLa cells with or without over-expression of wild-type alpha-synuclein (transfection − and +). Arrow indicates alpha-synuclein. B: Quantification of alpha-synuclein expression in the presence of various stimuli. DMSO (−), IL-1β (100 ng/mL), lipopolysaccharides (L2654 and L6529 1 µg/mL), βFGF (100 ng/mL), NGF (100 ng/mL) and dopamine (DA 50 µM) were added to 293 cells for 48 h and relative expression of alpha-synuclein was compared by quantitative RT-PCR. Relative amounts of alpha-synuclein mRNA were normalized to DMSO = 1.0, * P<0.01, ** P<0.001. C: Structure of the SNCA CpG island Entrez gene NC_000004.11 ID was analyzed by CpG island search software (http://www.uscnorris.com/cpgislands2/cpg.aspx.). Non-coding exon1 and coding exon2 are indicated by open arrows. The translation start ATG codon (methionine) located at exon2 is indicated by a closed circle. Two separate CpG islands are indicated by closed bars. D: Methylated-CpG island recovery assay of dopamine treated cells. 293 cells treated with or without dopamine were lysed and methylated. CpGs were enriched by MBD2 protein. Methylated CpG-1 or CpG-2 was detected by PCR before and after enrichment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991358&req=5

pone-0015522-g001: Identification of SNCA CpG islands.A: 293 cells express endogenous alpha-synuclein Immunoblot of cell lysates from SH-SY5Y, 293, and HeLa cells with or without over-expression of wild-type alpha-synuclein (transfection − and +). Arrow indicates alpha-synuclein. B: Quantification of alpha-synuclein expression in the presence of various stimuli. DMSO (−), IL-1β (100 ng/mL), lipopolysaccharides (L2654 and L6529 1 µg/mL), βFGF (100 ng/mL), NGF (100 ng/mL) and dopamine (DA 50 µM) were added to 293 cells for 48 h and relative expression of alpha-synuclein was compared by quantitative RT-PCR. Relative amounts of alpha-synuclein mRNA were normalized to DMSO = 1.0, * P<0.01, ** P<0.001. C: Structure of the SNCA CpG island Entrez gene NC_000004.11 ID was analyzed by CpG island search software (http://www.uscnorris.com/cpgislands2/cpg.aspx.). Non-coding exon1 and coding exon2 are indicated by open arrows. The translation start ATG codon (methionine) located at exon2 is indicated by a closed circle. Two separate CpG islands are indicated by closed bars. D: Methylated-CpG island recovery assay of dopamine treated cells. 293 cells treated with or without dopamine were lysed and methylated. CpGs were enriched by MBD2 protein. Methylated CpG-1 or CpG-2 was detected by PCR before and after enrichment.
Mentions: Using 293 cells, we tested various drugs that have been reported to alter alpha-synuclein expression. Of 6 reagents tested, 1 lipopolysaccharide and dopamine caused a significant increase in the production of alpha-synuclein (Figure 1B). Because dopamine seemed to enhance alpha-synuclein expression more than the lipopolysaccharide, we chose to analyze its effect on SNCA CpG island methylation.

Bottom Line: Gene multiplication can cause inherited PD, and promoter polymorphisms that increase SNCA expression are associated with sporadic PD.By using cultured cells, we identified a region of the SNCA CpG island in which the methylation status altered along with increased SNCA expression.This CpG region may function as an intronic regulatory element for SNCA gene.

View Article: PubMed Central - PubMed

Affiliation: Division of Neuroscience, Department of Neurology, Graduate School of Medicine, The University of Tokyo, Bunkyo, Tokyo, Japan.

ABSTRACT

Background: Alpha-synuclein (SNCA) gene expression is an important factor in the pathogenesis of Parkinson's disease (PD). Gene multiplication can cause inherited PD, and promoter polymorphisms that increase SNCA expression are associated with sporadic PD. CpG methylation in the promoter region may also influence SNCA expression.

Methodology/principal findings: By using cultured cells, we identified a region of the SNCA CpG island in which the methylation status altered along with increased SNCA expression. Postmortem brain analysis revealed regional non-specific methylation differences in this CpG region in the anterior cingulate and putamen among controls and PD; however, in the substantia nigra of PD, methylation was significantly decreased.

Conclusions/significance: This CpG region may function as an intronic regulatory element for SNCA gene. Our findings suggest that a novel epigenetic regulatory mechanism controlling SNCA expression influences PD pathogenesis.

Show MeSH
Related in: MedlinePlus