DNA methylation of the MAPT gene in Parkinson's disease cohorts and modulation by vitamin E in vitro

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder for which environmental factors influence disease risk and may act via an epigenetic mechanism. The microtubule-associated protein tau (MAPT) is a susceptibility gene for idiopathic PD. Methylation levels were determined by pyrosequencing of bisulfite-treated DNA in a leukocyte cohort (358 PD patients and 1084 controls) and in two brain cohorts (Brain1, comprising 69 cerebellum controls; and Brain2, comprising 3 brain regions from 28 PD patients and 12 controls). In vitro assays involved the transfection of methylated promoter-luciferase constructs or treatment with an exogenous micronutrient. In normal leukocytes, the MAPT H1/H2 diplotype and sex were predictors of MAPT methylation. Haplotype-specific pyrosequencing confirmed that the H1 haplotype had higher methylation than the H2 haplotype in normal leukocytes and brain tissues. MAPT methylation was negatively associated with MAPT expression in the Brain1 cohort and in transfected cells. Methylation levels differed between three normal brain regions (Brain2 cohort, putamen < cerebellum < anterior cingulate cortex). In PD samples, age at onset was positively associated with MAPT methylation in leukocytes. Moreover, there was hypermethylation in the cerebellum and hypomethylation in the putamen of PD patients compared with controls (Brain2 cohort). Finally, leukocyte methylation status was positively associated with blood vitamin E levels, and the effect was more significant in H2 haplotype carriers; this result was confirmed in cells that were exposed to 100 μM vitamin E. The significant effects of sex, diplotype, and brain region suggest that hypermethylation of the MAPT gene is neuroprotective by reducing MAPT expression. The effect of vitamin E on MAPT represents a possible gene-environment interaction.

Keywords: DNA methylation; Parkinson's disease; microtubule-associated protein tau; vitamin E.

Figure 1

Schematic diagram of the MAPT promoter region and CpG island. (A). Polymorphisms predicted to form CpG dinucleotides corresponding to either the H1 or H2 haplotype The region examined by the pyrosequencing assay (inverted triangle), and the polymorphism used to differentiate between H1 and H2 haplotype are indicated. Effect of MAPT diplotype and gender on MAPT promoter methylation in leukocyte DNA from (B) controls, (C) PD individuals. *** = p < 0.0001.

Figure 2

Haplotype-specific pyrosequencing of MAPT methylation. Methylation levels for the two haplotypes in each sample are indicated as paired data. Tissues examined were leukocyte, cerebellum, anterior cingulate cortex (ACC) and putamen. * = p < 0.05.

Figure 3

Effect of MAPT methylation on gene expression. (A) Scatter plot of MAPT methylation versus MAPT expression levels in cerebellum samples of Brain1 cohort (N = 69). Effect of MAPT methylation on PD. (B) In vitro analysis of relative expression in unmethylated (white) versus methylated (black) luciferase reporter constructs in SK-N-F1 cell. Error bars indicate standard error of the mean from five independent experiments. (C) Scatter plot of methylation versus PD age of onset from Leukocyte cohort (N=358). (D) Analysis of relative MAPT methylation in control (white, N = 12) and PD (black, N = 28) patients in cerebellum (W = 185, p = 0.034), anterior cingulate cortex (W = 210.5, p = 0.151) and putamen (W = 384.5, p = 0.001) from Brain2 cohort. Error bars indicate standard error of the mean. * = p < 0.05, ** = p < 0.001.

Figure 4

Effect of vitamin E on MAPT methylation. A + B) Scatter plots of vitamin E versus MAPT methylation levels in individuals segregated by their diplotype status as H1 (H1/H1 homozygotes) and H2 carriers (H1/H2 heterozygotes and H2/H2 heterozygotes). C) Dose response effect of vitamin E on either the H1 (open bar) or H2 (grey bar) of MAPT in SK-N-F1 cells. Methylation levels of each replicate were normalized against the value observed for the H1 haplotype at 0 mM vitamin E. Error bars indicate standard error of the mean from five independent experiments * = p < 0.05, ** = p < 0.001.