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) agonistd-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebrald-aspartate levels is due to the concomitant onset ofd-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylicd-amino acids. In the present work, we show thatd-aspartate content in the mouse brain drastically decreases after birth, whereasDdomRNA levels concomitantly increase. Interestingly, postnatalDdogene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control onDdoexpression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistentDdogene hypermethylation in the brain, we usedDdoknock-out mice (Ddo−/−), which show constitutively suppressedDdoexpression. In these mice, we found for the first time substantially increased extracellular content ofd-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation ofd-aspartate levels inDdo−/−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 inDdo−/−brains, highlights an unexpected importance ofDdodemethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of freed-aspartate. SIGNIFICANCE STATEMENTThe enzymed-aspartate oxidase (DDO) catalyzes the degradation of the NMDA receptor agonist,d-aspartate. In the brain, DDO is expressed only during postnatal life, thus reducing the embryonic storage ofd-aspartate and keeping thisd-amino acid at low levels during adulthood. Although the presence of DDO in mammals is long established, its biological role in the brain and the mechanism regulating its expression are still unclear. Here, we found thatDdopromoter demethylation enables the postnatal expression ofDdo. Moreover, persistent suppression ofDdoexpression leads to persistent spillover of extracellulard-aspartate and produces precocious cell death in the mouse brain, thus suggesting a key role for DDO in preventing early neurodegeneration triggered by excessive NMDA receptor stimulation.