Age-related changes in D-aspartate oxidase promoter methylation control extracellular D-aspartate levels and prevent precocious cell death during brain aging

The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain since it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that D-aspartate content in the mouse brain drastically decreases after birth while Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knockout mice (Ddo-/-), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of D-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo-/- brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo-/- brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by non physiological extracellular levels of free D-aspartate.