Alpha synuclein overexpression alters epigenetic regulation of neurodevelopmental and metabolic pathways in dopaminergic neurons Schaffner, S. L.1,2, Lazaro, D.3, Paiva, I.3, Outeiro, T. F.3,4,5, & Kobor, M. S.1,2,6 for Molecular Medicine and Therapeutics, BC Children’s Hospital, Vancouver, BC, Canada; 2Dept. of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada; 3Department of Experimental Neurodegeneration, Center for Nanoscale Microscopy and Molecular Physiology of the Brain, Center for Biostructural Imaging of Neurodegeneration, University Medical Center Goettingen, 37073, Goettingen, Germany 4CEDOC – Chronic Diseases Research Center, Faculdade de Ciencias Medicas, Universidade Nova de Lisboa, Lisboa, Portugal 5Max Planck Institute for Experimental Medicine, 37075 Goettingen, Germany 6Human Early Learning Partnership, University of British Columbia, Vancouver, BC, Canada 1Centre D In this study, we investigated the effects of SNCA overexpression and mutation on two epigenetic marks – DNA methylation and DNA hydroxymethylation – in a dopaminergic neuron tissue culture system, using the Illumina EPIC BeadChip Array. We found that wild-type and A30P mutant SNCA had many similar effects on the DNA methylome of dopaminergic neurons. However, each SNCA genotype also had distinct effects on the epigenome; the wild type protein altered DNA methylation at more CpG sites overall, found in neurodevelopmental and synaptic function genes, while the A30P mutant altered DNA methylation and hydroxymethylation at CpG sites found in genes regulating glucose metabolism. 3.1. Results: Both SNCA-OE and A30P cells were differentially methylated at genes involved in motor function. D F 1. Introduction A-B) Volcano plots showing results of linear modeling (DNAhm ~ Genotype) in control vs SNCA-OE or A30P cells; red = sites with decreased DNAm at FDR <= 0.05 and absolute delta beta >= 0.05; grey = non-significant sites; blue = sites with decreased DNAm at FDR <= 0.05 and absolute delta beta >= 0.05. C) Venn diagram showing the number of differentially hydroxymethylated sites unique to each genotype and shared between genotypes. D) Heatmap showing beta values at the 37 CpG sites that were differentially hydroxymethylated in A30P cells only; red = low DNAm, blue = high DNAm. E) Top three CpG sites that were differentially hydroxymethylated in A30P cells only, ranked by delta beta. F) Result of gene ontology enrichment analysis on the 37 CpG sites differentially hydroxymethylated in A30P cells only at p < 0.01. 3.5. Results: DNA methylation and hydroxymethylation may contribute to transcriptional deregulation of a glutamate signaling network in both SNCAOE and A30P cells. A NS expression N NoData N D Up 0 1.39>5.99 edge C24 D N N I N D Wang et al., Front Mol Neurosci 2016 • SNCA mutation/multiplication alone doesn’t always lead to familial PD; and SNCA SNPs also contribute to sporadic PD2 • The epigenome may mediate PD genetic risk from SNCA and environmental risk from lifestyle, nutrition, and toxic exposures3-6 • DNA methylation and DNA hydroxymethylation are two stable, easily measurable epigenetic marks in brain tissue which may influence gene expression7-8 A) Heatmap showing beta values at a sample of 100 CpG sites that were differentially methylated in SNCA-OE cells only; red = low DNAm, blue = high DNAm. B) Top three CpG sites that were differentially methylated in SNCA-OE cells only, ranked by delta beta. C) Result of gene ontology enrichment analysis on the 2428 CpG sites differentially methylated in SNCA-OE cells only at p < 0.01. A) Heatmap showing beta values at the 24 CpG sites that were differentially methylated in A30P cells only; red = low DNAm, blue = high DNAm. B) Top three CpG sites that were differentially methylated in A30P cells only, ranked by delta beta. C) Result of gene ontology enrichment analysis on the 24 CpG sites differentially methylated in A30P cells only at p < 0.01. Williams et al., EMBO Reports 2012 Lill, Mol Cell Probes 2016 OpenSea/Body SNCA-OE DB -0.08 SNCA-OE FDR 0.04 DLG4 C C DNAm hits (FDR <= 0.01, DB >= 0.1) GRIK1 CpG island/TSS200 SNCA-OE DB -0.102 SNCA-OE FDR 1.87e-7 GRIK2 DLG3 CpG island/TSS200 SNCA-OE DB -0.135 SNCA-OE FDR 2.45e-7 Paiva et al., Hum Mol Genet 2017 A30P DB -0.05 A30P FDR 0.03 GRIK4 DLG1 CpG island/TSS200 SNCA-OE DB -0.102 SNCA-OE FDR 1.54e-6 OpenSea/Body SNCA-OE DB 0.139 SNCA-OE FDR 1.76e-7 GRIK5 A) Regulatory protein-protein interaction module generated using DNA methylation, DNA hydroxymethylation, and gene expression changes between control and SNCAOE cells. B) Differentially hydroxymethylated CpG site within the GRIK2 gene. C) Differentially methylated CpG sites within the GRIK2 gene. D) GRIK2 expression fold change in control, SNCA-OE, and A30P cells. Conclusions • • B • • 3.3. Results: A30P cells were differentially methylated at genes involved in glucose metabolism. GRIK3 NCALD A-B) Volcano plots showing results of linear modeling (DNAm ~ Genotype) in control vs SNCA-OE or A30P cells; red = sites with decreased DNAm at FDR <= 0.01 and absolute delta beta >= 0.1; grey = non-significant sites; blue = sites with decreased DNAm at FDR <= 0.01 and absolute delta beta >= 0.1. C) Venn diagram showing the number of differentially methylated sites unique to each genotype and shared between genotypes. D) Heatmap showing beta values at the 129 CpG sites that were differentially methylated in both genotypes; red = low DNAm, blue = high DNAm. E) Top three CpG sites that were differentially methylated in both genotypes, ranked by delta beta. F) Result of gene ontology enrichment analysis on the 129 CpG sites differentially methylated in both genotypes at p < 0.01. A Glutamate receptor I hydroxy promoter Guanylate cyclase; NMDA receptor clustering at synapses 3.2. Results: SNCA-OE cells were differentially methylated at genes involved in neurodevelopment and synaptic function. D hydroxy body methylation enhancer E Kara et al., Neurosci Letters 2013 DNAhm hit (FDR <= 0.05, DB => 0.05) D 0.02 5.37>9.49 methylation body B Network built around GRIK2 Chi−square P−value= 0 hydroxy enhancer N D N I I 2 I methylation promoter Down N node C2 N • Under pathological conditions or when overexpressed, SNCA can form oligomers, fibrils, and aggregates, impairing function1 • The A30P missense mutation disrupts the ability of SNCA to bind membranes1 N D N These results suggest that there are some epigenetic pathways that may be commonly dysregulated among those with different SNCA variants, and some that are unique. This may help in understanding why PD manifests differently across individuals, and encourages stratification by SNCA genotype for further research. F E I The alpha synuclein (SNCA) gene encodes a protein that is found at synaptic terminals and regulates neuronal differentiation, survival, and metabolism as well as SNARE complex assembly. Multiplications of SNCA and missense mutations are linked to familial Parkinson’s disease (PD). SNPs in SNCA may also contribute to development of sporadic PD. However, it is not fully understood why only some individuals with SNCA variants will develop disease, or what specific role SNCA SNPs play in sporadic PD. Environment and lifestyle likely modulate the probability that genetically at-risk individuals will develop PD, and the epigenome may mediate these genetic and environmental contributions to disease. 3.4. Results: A30P cells also had increased DNA hydroxymethylation at genes regulating glucose metabolism. 2. Methods N Abstract A C B Overexpression of wild type and A30P mutant SNCA have many similar effects on the epigenome, and may induce epigenetic and transcriptional dysregulation of motor function pathways such as GRIK2-DLG3 glutaminergic signaling Overexpression of wild type SNCA induces differential DNA methylation at genes regulating neurodevelopment and synaptic function Overexpression of A30P SNCA induces differential DNA methylation and hydroxymethylation at genes regulating glucose metabolism Ability of wild type vs mutant SNCA to bind membranes and/or receptor targets may underlie differential effects of each genotype on DNA methylation Acknowledgements Thank you to Julia MacIsaac, David Lin, and Katia Ramadori for running samples on the EPIC BeadChip. Thank you also to Rachel Edgar and David Lin for advice on analysis of DNA hydroxymethylation data. SLS was supported by a Faculty of Medicine Four Year Doctoral Fellowship. MSK is the Canada Research Chair in Social Epigenetics, Senior Fellow of the Canadian Institute for Advanced Research, and Sunny Hill BC Leadership Chair in Child Development. This work is conducted under a Transnational “Epigenomics of Complex Diseases” grant (CIHR EGM-141897, ANR-15-EPIG-0001, BMBF 01KU1503A). References 1Wang, C. et al. Fron Mol Neurosci 2016, 9:48. 2Lesage, S., & Brice, A. Hum Mol Genet 2009, R48-R59. 3Ascherio, A., & Schwarzchild, M. A. Lancet Neurol 2016, 14:1257-1272. 4Coppede, F. Sci World J 2012, 489830. 5Chuang, Y., et al. Genome Med 2017, 9:76. 6Wassouf, Z., et al. Front Cell Neurosci 2018, 12:112. 7Jones, P. A. Nat Rev Genet, 13: 484-492. 8Wen, L., Li, X., Yan, L. et al. Genome Biol 2014, 15(3):1-17.