Alpha-synuclein overexpression is associated with epigenomic dysregulation of glutamate signaling and locomotor pathways

Although Parkinson’s disease (PD) is one of the most rapidly growing neurological disorders, interindividual differences in PD etiology related to genetics are not fully understood. Here, we demonstrate genome-wide DNA methylation and hydroxymethylation alterations associated with overexpression of two PD-linked alpha-synuclein variants (wild type and A30P) in LUHMES cells differentiated to dopaminergic neurons. Alpha-synuclein altered DNA methylation at thousands of CpGs and DNA hydroxymethylation at hundreds of CpGs in both genotypes, and primarily at locomotor and glutamate signaling pathway genes. In some cases, epigenetic changes were associated with transcription. SMITE network analysis incorporating H3K4me1 ChIP-seq to score DNA methylation and hydroxymethylation changes across promoters, enhancers, and gene bodies confirmed epigenetic and transcriptional deregulation of glutamate signaling modules in both genotypes. Our results identify distinct and shared impacts of alpha-synuclein variants on the epigenome, and associate alpha-synuclein in the epigenetic etiology of PD. DNA from Lund Human Mesencephalic (LUHMES) cells was used to assess the impact of alpa-synuclein (aSyn) on cellular DNA methylation and hydroxymethylation. For this purpose, three different cell lines were generated. Briefly, proliferating LUHMES cells were infected with equimolar amounts of lentiviral particles encoding for: IRES-GFP (samples set C); full-length human wild-type (WT) aSyn (SNCA, NM_000345), WT aSyn-IRES-GFP (samples set E) or familial mutant A30P aSyn, A30P aSyn-IRES-GFP (samples set A). Positive green fluorescent cells were selected by fluorescence activated cell sorting. Seven independent replicates were generated for the GFP (control) cell line and eight independent replicates were generated for the WT and A30P aSyn lines. Total DNA from differentiated LUHMES cells, at differentiation day 8, was extracted and purified using the AllPrep DNA/RNA Mini Kit (Qiagen), according to the instructions of the manufacturer. DNA quantity and purity were assessed by spectrophotometry. 1.5 μg of DNA per sample was split into two 750 ng aliquots for sodium bisulfite (BS) conversion or oxidative bisulfite (oxBS) conversion using the NuGEN TrueMethyl oxBS Module (NuGEN Technologies Inc.). One aliquot per sample was treated with the oxidation protocol while the second aliquot underwent mock oxidation; all samples were then sodium bisulfite converted according to the manufacturer’s instructions.