5-Hydroxymethylcytosine, the "Sixth Base", during brain development and ageing.

The epigenome is of fundamental importance for development and ageing. The discovery of 5-hydroxymethylcytosine (5hmC), a further base modification of cytosine beyond 5-methylcytosine, might be of high relevance in understanding the complexity of the human brain, as 5hmC is found in great extent in brain tissue. The aim of this study was to investigate the quantity of 5hmC containing nuclei by immunohistochemistry in human and murine brains at several developmental stages. We performed immunohistochemical stainings on frontal cortex, white matter and cerebellar cortex of 15 healthy controls. Three cases each were assigned to five age groups (foetus, adolescent, adult, elderly, aged). Additionally, cortex and cerebellum of 15 mice sacrificed between day 0 and 120 after birth were investigated. We found marked alterations of 5hmC amount during ageing. In human cortex there was an increase of 5hmC of 50%, in white matter we found an increase of even 200% during ageing. In the cerebellum both internal granular cell layer and molecular cell layer showed a significant increase of 5hmC till adulthood. Purkinje cell nuclei showed constantly positive signals for 5hmC. These data were paralleled in murine brains. Co-labelling of 5hmC and markers for mature and immature cells in murine cerebellar cortex at the age of 7 days revealed that 5hmC was found in mature but not in immature cells. In conclusion, the findings described in this study emphasise the importance of 5hmC in brain development and ageing and will help to better understand the complexity and plasticity of the brain.