On the basis of these reports, we hypothesized that there may be another unknown mechanism for increased SNCA expression in PD. In addition to promoter polymorphisms, epigenetic modification can alter downstream gene expression. Epigenetic regulation includes histone modification and DNA methylation, of which CpG island methylation can be gene-specific; in several different cancers, CpG methylation inhibits binding of the transcription machinery, causing silencing of a specific oncogene, which leads to carcinogenesis. In central nervous system disorders, CpG methylation has been associated with psychiatric disorders, such as autism and schizophrenia. We sought to identify CpG islands in the SNCA gene,Demethylzeylasteral wherein methylation status was associated with alpha-synuclein expression. For this purpose, we had to find cell lines which express endogenous alpha-synuclein at sufficient levels to support a comparative analysis. We analyzed the effects of several reagents on alpha-synuclein expression in 293 cells and found that dopamine had the most pronounced effect without affecting cell viability. We also tried to screen reagents that altered CpG methylation along with increased alpha-synuclein expression level in SH-SY5Y cells; however, reagents we used did not increase its expression level without affecting cell viability significantly. To avoid the possibility of CpG demethylation by cell death, we decided to use 293 cells instead of SH-SY5Y cells. Using dopamine as a modulator of SNCA expression, we searched for a CpG island which exhibited altered methylation which correlated with changes in gene expression. Of 2 CpG islands identified in the SNCA gene, we located the second CpG island at the first intronic region, and found that it showed a significant decrease in Epimedin-B methylation, associated with induction of alpha-synuclein expression. This was first identified by MIRA method and was confirmed by bisulfite sequencing, which yielded comprehensive results. We found that 293 cells expressed D1 and D2 receptors, which supported the rationale for our investigation of dopaminemediated CpG demethylation in this cell line although they are not neuronal cells. Additional experiments showed that haloperidol, the D2 receptor antagonist, clearly inhibited demethylation by dopamine; thus we concluded that this demethylation was mediated by dopamine receptor D2. Previous studies have compared alpha-synuclein expression in postmortem brain or peripheral blood from PD patients, with inconsistent results, partly because of artifacts of sample quality and degradation of RNA extracts from postmortem brain tissue. However, the result from substantia nigra was quite striking; the methylation level was almost zero in the PD group, whereas in the control group it was almost 100%, suggesting that methylation in the substantia nigra is an important component of PD pathogenesis. For concerns about DNA quality from substantia, nigra, we tested in several ways to assure that those were comparable between controls and PDs. The demethylatyion of CpG-2 could be occurring in both neurons and glial cells especially because in the late disease course, massive neuronal loss can occur. However, this does not indicate that our findings is unrelated to the direct pathogenesis, since it has widely recognized that alpha-synuclein aggregation are observed also in the astrocytes and oligodendrocytes of PD brains. In neurodegenerative diseases, it is still a mystery that neurons in similar functions start to degenerate simultaneously regardless of their spatial location in the brain. We can speculate that in the early developmental stage, even before the neuronal and the glial cells are differentiated, epigenetical abnormality can occur.