Genome-Wide Profiling of DNA Methylation and Gene Expression induced by Alpha-Synuclein in Murine Microglia [methylation]

BACKGROUND: Synucleinopathy disorders are characterized by aggregates of α-synuclein (α-syn), which engage microglia to elicit a neuroinflammatory response. However, the influence α-syn has on DNA methylation and gene expression patterns for other genes has not been thoroughly explored. Our objective is to determine gene expression and methylation changes in microglia induced by aggregate α-syn.METHODS: Transgenic mThy1-Asyn mice overexpressing human α-syn are a model of synucleinopathy. Microglia from 3 and 13-month-old mice were used to isolate nucleic acids for methylated DNA and RNA-sequencing. α-Syn-induced changes in gene expression and genomic methylation were determined and examined for functional enrichment followed by network analysis to further elucidate possible connections within the data.RESULTS: Microglial DNA isolated from our 3-month cohort had 5,315 differentially methylated gene (DMG) changes, while RNA levels demonstrated a change in 119 differentially expressed genes (DEGs) between mThy1-Asyn mice and wild-type littermate controls. The 3-month DEGs and DMGs were highly associated with adhesion and migration signaling, suggesting a phenotypic transition from resting to active microglia. We observed 3,742 DMGs and 3,766 DEGs in 13-month mThy1-Asyn mice.These genes were often related to adhesion, migration, cell cycle, cellular metabolism, and immune response. Network analysis also showed increased cell mobility and inflammatory functions at 3 months, shifting to cell cycle, immune response, and metabolism changes at 13 months.CONCLUSION: We observed significant α-syn-induced methylation and gene expression changes in microglia. Our data suggest that α-syn overexpression initiates microglial activation leading to neuroinflammation and cellular metabolic stresses, which is associated with disease progression. We performed transcriptomic and methylomic analysis of murine brain homogenate subjected to microglia selection enrichment from mice overexpressing full-length human wild-type α-syn under the murine Thy-1 promoter on the X chromosome. Transcriptomic and methylomic profiles were analyzed for biological meaning using Reactome pathway database, Gene ontology, and Kyoto Encyclopedia of Genes and Genomes on differentially expressed genes (DEGs) and differentially methylated genes (DMGs). Pathways were also examined by enrichment network anlaysis and MCODE to identify clusters of highly connected pathways. HEM samples are mice overexpressing human alpha-synuclein while WT mice are controls. The corresponding background of these mice is the B6D2F1/J mice (stock# 100006) from The Jackson Laboratory (Bar Harbor, ME). There are eight 13 month old HEM mice and four 13 month wild-type controls. There are four 3 month HEM mice and three 3month wild-type controls.