Methylation regulates alpha-synuclein expression and is decreased in Parkinson's disease patients' brains

Abstract

Alpha-synuclein (SNCA) is a major risk gene for Parkinson's disease (PD), and increased SNCA gene dosage results in a parkinsonian syndrome in affected families. We found that methylation of human SNCA intron 1 decreased gene expression, while inhibition of DNA methylation activated SNCA expression. Methylation of SNCA intron 1 was reduced in DNA from sporadic PD patients' substantia nigra, putamen, and cortex, pointing toward a yet unappreciated epigenetic regulation of SNCA expression in PD.

Figure 1.

The SNCA region of interest. a, Schematic drawing of the 5′ region of SNCA with exons 1 and 2 (boxes) and the 5′ UTR and intron 1 (line). The arrow indicates the position of the putative core promoter; the triangle indicates the position of a rudimentary TATA box. Dimension of the CGI is depicted by the stripped box. Numbers are relative to the translation start site (ATG + 1). b, Fragments SNCA_{(−2079/−1507)} (promoter) and SNCA_{(−926/−483)} (intron) were used for bisulfite sequencing. c, Fragment SNCA_{(−1524/−189)} was used for reporter assays. d, Predicted transcription factor binding sites within SNCA_{(−926/−483)} (intron) adjacent to CpG dinucleotides.

Figure 2.

Status of SNCA methylation in SK-N-SH cells and impact on expression. a, SK-N-SH cells were incubated with DMSO [control (Ctrl)] or 10 μm Aza for 24 and 48 h, respectively, and isolated DNA was used for bisulfite sequencing of SNCA_{(−2079/−1507)} (promoter) and SNCA_{(−926/−483)} (intron); 10 independent clones for each condition were analyzed by bisulfite sequencing. The lollipop diagram with unmethylated CpG sites (open circles) and methylated CpG sites (closed circles) illustrates the higher degree of methylation in SNCA_{(−926/−483)} (intron) compared with SNCA_{(−2079/−1507)} (promoter) and the decrease of methylated CpG in SNCA_{(−926/−483}) (intron) after addition of Aza. b, After 48 h of Aza treatment, the fraction of methylated CpGs is reduced by ∼60% in SNCA_{(−926/−483)} (intron). c, Treatment with Aza induced a time-dependent increase of SNCA expression exceeding 50% of Ctrl. Quantitative RT-PCR was performed in triplicate and presented as the mean ± SD. d, Representative Western blot with anti-synuclein antibody demonstrates an increase of synuclein protein after addition of Aza too.

Figure 3.

Promoter activity of SNCA_{(−1524/−189)} in HeLa cells. In vitro methylation of the reporter construct SNCA_{(−1524/−189)} reduced relative luciferase activity by 98.5% (**p ≤ 0.01, one-way ANOVA). Absence of SssI or SAM from the in vitro methylation reaction minimized reduction. Ctrl, Construct with the inverted insert.

Figure 4.

Status of SNCA_{(−926/−483)} methylation in human brain. a, Mean percentages (±SD) of methylated CpG sites summed up over all 23 CpG sites analyzed. PD patients' DNA was hypomethylated compared with the neurologically healthy individuals in all analyzed brain regions. *p ≤ 0.05, **p ≤ 0.01. b, Detailed comparison of the methylation of SNCA_{(−926/−483)} in DNA from SNpc, putamen, and cortex of PD and neurologically healthy individuals. Percentage of methylated CpG at the particular position in the three different brain regions. Asterisks indicate significant (p ≤ 0.05, one-way ANOVA) hypomethylation in PD patients compared with control (mean ± SD of at least 10 clones per individual per CpG position analyzed by bisulfite sequencing).