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

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.

Figure 1. 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.

Figure 2. Dopamine induced demethylation of CpG-2.

A: Location of CpGs in the second SNCA CpG island. There are 13 CpGs in the CpG-2 region. Each CpG is labeled above according to its positions from the 5′ end of the sequence. GATA transcription factor recognition sequence is shown with an under bar. B: Bisulfite sequence analysis of the CpG-2 region. Dopamine (DA; 0, 50, and 100 µM) was added to 293 cells. DNA was purified and bisulfite sequence analysis was performed. At least 20 clones from each dopamine-treated group were analyzed. Open circles indicate unmethylated CpGs, whereas closed circles indicate methylated CpGs. The degree of methylation was calculated as methylated CpG/total CpG and shown below (% methylation).

Figure 3. CpG-2 demethylation up-regulates SNCA expression through dopamine receptor signaling.

A: Dose dependent up-regulation of alpha-synuclein by dopamine. RNA was extracted from cells treated with the indicated dopamine concentration for 48 h and quantitative PCR was performed. The relative amount of alpha-synuclein mRNA against beta-actin was normalized to dopamine = 0. * P<0.05 vs. DA = 0. B: Dopamine does not affect cell viability. MTS assay was performed after addition of dopamine for 48 h. P = 0.1559 with ANOVA. C: Methylation of CpG-2 suppresses downstream gene expression. Luciferase assays were performed after transfection of a CpG-free firefly luciferase vector harboring the CpG-2 region at the 5′ of the luciferase gene. The vector was treated with (+) or without (-) CpG methyltransferase prior to transfection. Sea pansy luciferase was co-transfected as an internal control. Relative luciferase activity was calculated by dividing firefly luciferase activity by sea pansy luciferase activity and normalized to methylation (−) as 100%. Bars indicate standard error. * P = 0.0025. D: 293 cells express D1 and D2 dopamine receptors. Expression of DRD1, −2 and −3 were analyzed in HCN-2, SH-SY5Y, and 293 cells by quantitative RT-PCR. Relative amounts of each receptor are normalized to HCN-2 = 1. E: A D2 receptor antagonist ameliorates the demethylation effect of dopamine. Dopamine (DA)- and haloperidol-treated 293 cells were lysed and methylated CpGs were enriched by MBD2 protein. Methylated CpG-2 was detected by PCR before and after enrichment.

Figure 4. Analysis of CpG-2 methylation level in various brain regions.

A–C: CpG-2 methylation was analyzed by bisulfite sequencing and plotted against age. Data from anterior cingulate cortex (control: n = 8, PD/DLB: n = 12) (A), putamen (control: n = 4, PD/DLB: n = 7) (B), and substantia nigra (control; n = 3, PD/DLB: n = 4) (C) are shown. Closed circle = controls, open box = PD/DLB. In figure 4C, age and % methylation had no significant correlation P = 0.2321. D–F: CpG-2 methylation was analyzed by bisulfite sequencing method and plotted by disease groups. Data from anterior cingulate cortex (D), putamen (E) and substantia nigra (F) are shown. Closed circle = controls, open box = PD, closed box = DLB P<0.0001 vs. control.

Figure 5. Specific CpG-2 demethylation in substantia nigra of Parkinson's disease patients.

CpG-2 methylation in the anterior cingulate cortex (A), putamen (B), and substantia nigra (C) of PD (open boxes) and DLB (closed boxes) are plotted against disease duration.