342 - Ascorbate supplementation increases 5-hydroxymethylcytosine in brain cerebellum DNA of ascorbate knockout mice

Abstract

5-Hydroxymethylcytosine (5hmC) is a newly characterized epigenetic mark arising from the enzymatic oxidation of 5-methylcytosine (5mC) in DNA by the ten eleven translocation (TET) family enzymes. Whereas 5mC supresses gene expression, the presence of 5hmC correlates with the upregulation of certain genes. Using LC-MS/MS, we compared the global levels of genomic 5mC, 5hmC and 5-formylcytosine (5fC) in the brains of 4-month-old wild-type mice on a standard diet without ascorbate in comparison with Gulo-/- ascorbate knockout mice on a standard diet plus 0, 0.1, and 4.0 g/L ascorbate supplements. In addition, these modifications were measured in 20-month-old wild-type mice. Four regions of the brain were examined, which included cerebellum, cortex, hippocampus and striatum. The total brain weight and particularly the cerebellum weight of Gulo-/- mice were lower than those of wild-type mice. There was also a reduction of both epigenetic marks (5mC and 5hmC) in the cerebellum of Gulo-/- compared to wild-type mice. In contrast, weight, 5mC, 5hmC did not significantly change in other regions of the brain between the age-matched ascorbate deficient Gulo-/- mice. Loss of 5hmC in the cerebellum was increased in going from extreme deficiency (no ascorbate) to ascorbate supplements (4.0 g/L) in Gulo-/- mice. Lastly, similar decreases in weight and the level of 5mC and 5hmC were observed with age when comparing 4-month-old to 20-month-old wild-type mice. These results demonstrate that ascorbate modulates 5hmC in vivo and that the cerebellum is the most sensitive region affected by brain ascorbate deficiency. This work links dietary ascorbate to the epigenetic regulation of pathways that may be involved in a variety of neurological disorders.