Multiple system atrophy (MSA) is a fatal late-onset neurodegenerative disease. Although presenting with distinct pathological
hallmarks, which in MSA consist of glial cytoplasmic inclusions (GCIs) containing fbrillar α-synuclein in oligodendrocytes, both MSA and Parkinson’s disease are α-synucleinopathies. Pathologically, MSA can be categorized into striatonigral
degeneration (SND), olivopontocerebellar atrophy (OPCA) or mixed subtypes. Despite extensive research, the regional
vulnerability of the brain to MSA pathology remains poorly understood. Genetic, epigenetic and environmental factors have
been proposed to explain which brain regions are afected by MSA, and to what extent. Here, we explored for the frst time
epigenetic changes in post-mortem brain tissue from MSA cases. We conducted a case–control study, and profled DNA
methylation in white mater from three brain regions characterized by severe-to-mild GCIs burden in the MSA mixed subtype
(cerebellum, frontal lobe and occipital lobe). Our genome-wide approach using Illumina MethylationEPIC arrays and a
powerful cross-region analysis identifed 157 CpG sites and 79 genomic regions where DNA methylation was signifcantly
altered in the MSA mixed-subtype cases. HIP1, LMAN2 and MOBP were amongst the most diferentially methylated loci. We
replicated these fndings in an independent cohort and further demonstrated that DNA methylation profles were perturbed
in MSA mixed subtype, and also to variable degrees in the other pathological subtypes (OPCA and SND). Finally, our comethylation network analysis revealed several molecular signatures (modules) signifcantly associated with MSA (disease
status and pathological subtypes), and with neurodegeneration in the cerebellum. Importantly, the co-methylation module
having the strongest association with MSA included a CpG in SNCA, the gene encoding α-synuclein. Altogether, our results
provide the frst evidence for DNA methylation changes contributing to the molecular processes altered in MSA, some of
which are shared with other neurodegenerative diseases, and highlight potential novel routes for diagnosis and therapeutic
interventions