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. 2024 Oct;61(10):7830-7844.
doi: 10.1007/s12035-024-03974-3. Epub 2024 Mar 2.

The Alpha-Synuclein Gene (SNCA) is a Genomic Target of Methyl-CpG Binding Protein 2 (MeCP2)-Implications for Parkinson's Disease and Rett Syndrome

Ina Schmitt #  1   2 , Bernd O Evert #  1 , Amit Sharma #  3 , Hassan Khazneh  1 , Chris Murgatroyd  4 , Ullrich Wüllner  5   6   7
Affiliations

The Alpha-Synuclein Gene (SNCA) is a Genomic Target of Methyl-CpG Binding Protein 2 (MeCP2)-Implications for Parkinson's Disease and Rett Syndrome

Ina Schmitt et al. Mol Neurobiol. 2024 Oct.

Abstract

Mounting evidence suggests a prominent role for alpha-synuclein (a-syn) in neuronal cell function. Alterations in the levels of cellular a-syn have been hypothesized to play a critical role in the development of Parkinson's disease (PD); however, mechanisms that control expression of the gene for a-syn (SNCA) in cis and trans as well as turnover of a-syn are not well understood. We analyzed whether methyl-CpG binding protein 2 (MeCP2), a protein that specifically binds methylated DNA, thus regulating transcription, binds at predicted binding sites in intron 1 of the SNCA gene and regulates a-syn protein expression. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility-shift assays (EMSA) were used to confirm binding of MeCP2 to regulatory regions of SNCA. Site-specific methylation and introduction of localized mutations by CRISPR/Cas9 were used to investigate the binding properties of MeCP2 in human SK-N-SH neuroblastoma cells. The significance of MeCP2 for SNCA regulation was further investigated by overexpressing MeCP2 and mutated variants of MeCP2 in MeCP2 knockout cells. We found that methylation-dependent binding of MeCP2 at a restricted region of intron 1 of SNCA had a significant impact on the production of a-syn. A single nucleotide substitution near to CpG1 strongly increased the binding of MeCP2 to intron 1 of SNCA and decreased a-syn protein expression by 60%. In contrast, deletion of a single nucleotide closed to CpG2 led to reduced binding of MeCP2 and significantly increased a-syn levels. In accordance, knockout of MeCP2 in SK-N-SH cells resulted in a significant increase in a-syn production, demonstrating that SNCA is a genomic target for MeCP2 regulation. In addition, the expression of two mutated MeCP2 variants found in Rett syndrome (RTT) showed a loss of their ability to reduce a-syn expression. This study demonstrates that methylation of CpGs and binding of MeCP2 to intron 1 of the SNCA gene plays an important role in the control of a-syn expression. In addition, the changes in SNCA regulation found by expression of MeCP2 variants carrying mutations found in RTT patients may be of importance for the elucidation of a new molecular pathway in RTT, a rare neurological disorder caused by mutations in MECP2.

Keywords: SNCA; Alpha-synuclein; DNA methylation; Epigenetic; Genomic target; Intron; MeCP2; Methyl-CpG binding protein 2; Parkinson’s disease; RTT; Rett syndrome.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1.
Fig. 1.
SNCA gene regions analyzed for DNA methylation and MeCP2 binding. A Schematic of the SNCA promoter (arrow) and exons 1 (E1) and 2 (E2). Position of the CpG island (CGI) is depicted by the striped box (adapted from 13). B Position of the SNCA(-926/-483) region containing CpGs 1-23 analyzed by bisulfite sequencing. C Position of the SNCA(-1524/-189) region used for reporter assays. Arrows indicate the position of restriction sites for the enzymes BstXI, BamHI, and HpaI. D PCR-amplified regions within SNCA intron 1 analyzed by ChIP assays with an MeCP2 antibody (ChIP1, -964/-792; ChIP2, -811/-617). E Sequence of SNCA(-1524/-189) showing CpGs in blue, CpGs 1-23 as part of SNCA(-926/-483) in black bold capitals. Sequence of ChIP1 (CpGs 1-7) is marked in red and pink, sequence of ChIP2 (CpGs 6-13) in pink and blue letters. [A/T] ≥ 4 motifs adjacent to CpGs are marked in green; recognition sites for the restriction enzymes BstXI, BamHI, and HpaI are underlined, and target sequences for CRISPR/Cas9 editing are highlighted in yellow boxes
Fig. 2
Fig. 2
Impact of SNCA methylation on MeCP2 binding. A Methylation status of SNCA(-926/-483) in SK-N-SH cells untreated (Ctrl) and treated with 10 μM Aza for 48 h (Aza). The degree of methylation is presented as the average percentage determined from 10 independent clones (n = 10; ± s.d.) by bisulfite sequencing in either the untreated and treated SK-N-SH cells. B Enrichment of SNCA fragments ChIP1 and ChIP2 for MeCP2 binding in Ctrl and Aza-treated SK-N-SH cells. Data are presented as means ± s.d. of three independent ChIP reactions. C Representative Western blot analysis of MeCP2 and actin expression in SK-N-SH cells treated with Aza (0, 2.5, 5.0, and 10.0 μM) for 48 h. D Densitometric quantification of the Western blot results for MeCP2 protein levels. The values are expressed in relation to the respective actin levels. The results were averaged from triplicates of three independent experiments and are presented as the mean ± s.d. E Methylation status of SNCA intron 1 in human brain cortex from two healthy control (Ctrl) and two PD cases (PD). F MeCP2 binding to SNCA fragments ChIP1 and ChIP2 using ChIP analysis of human cortex samples from two controls (Ctrl) and 2 PD patients (PD)
Fig. 3
Fig. 3
A Schematic representation of site-specific methylation (asterisk); for details see “Materials and Methods”. B Degree of SNCA methylation in the vector construct after transfection into HeLa cells; 10 independent clones were used for bisulfite sequencing of SNCA(-926/-483). The lollipop diagram illustrates unmethylated CpGs by open circles and methylated CpGs by closed circles. Left: unmethylated BstXI/HpaI fragment. Middle: site-specific methylation of the BstXI/BamHI fragment. Right: site-specific methylation of the BamHI/HpaI fragment. C Luciferase activity after in vitro methylation of the entire SNCA(-1524/-189) construct (vector) and after site-specific methylation of the BstXI/BamHI fragment (CpGs 1-2) and the BamHI/HpaI-fragment (CpGs 4-9). Data are presented as means ± s.d. of six independent luciferase measurements. (*) p ≤ 0.05; (**) p ≤ 0.01
Fig. 4
Fig. 4
CRISPR/Cas9-mediated mutations in intron 1 of SNCA affect the expression of a-syn and binding of MeCP2. A Representative Western blot of a-syn and actin using lysates of the wild type (wt) and mutated SK-N-SH cell lines, C-841d and T-886G. B Quantification of three individual Western blots by densitometry. Relative protein amounts of a-syn were normalized to actin and compared to untreated SK-N-SH (wt). C Enrichment of MeCP2 to SNCA-fragments ChIP1 and ChIP2 using chromatin from cell lines C-841d and T-886G by ChIP analysis. Data are presented as means ± s.d. of three independent ChIP reactions. (*) p ≤ 0.05; (**) p ≤ 0.01
Fig. 5
Fig. 5
Effects of MeCP2 knockout and protein variants on a-syn expression. A Western blot analysis of MeCP2, a-syn and actin in lysates from native SK-N-SH cells (SK) and MeCP2 knockout cell lines (ko 1 and ko 2) transfected without/with (±) pCMV3-MeCP2. B Densitometric quantification of a-syn protein levels normalized to actin. C Schematic of the MeCP2 domain structure and localization of analyzed MeCP2 mutations. MBD, methyl-CpG binding domain; ID, intervening domain; TRD, transcriptional repression domain. D Western blot analysis of MeCP2, a-syn and actin in lysates from MeCP2 knockout cells either not transfected (/) or transfected with wildtype MeCP2 and different MeCP2 variants (wt and variants 140, 158, 255, and 270). E Densitometric quantification of a-syn and MeCP2 protein levels normalized to actin in comparison to untransfected MeCP2 knockout cells (/). Data are presented as means ± s.d. from five independent transfections. (*) p ≤ 0.05; (**) p ≤ 0.01
Fig. 6
Fig. 6
Binding properties of MeCP2 variants to CpG1 and CpG2 in SNCA intron 1. A EMSAs analyzing the binding to methylated SNCA probes CpG1 and CpG2 with increasing protein amounts of MeCP2 (30-75 nmol). (-) indicates free probe. B Densitometric quantification of signal intensities. Data are presented as relative shift signal normalized to the signal obtained without protein
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