Age-Related Changes in d-Aspartate Oxidase Promoter Methylation Control Extracellular d-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging

Abstract

The endogenous NMDA receptor (NMDAR) agonist D-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 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, whereas 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 knock-out mice (Ddo Ϫ/Ϫ ), 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 Ϫ/Ϫ 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 Ϫ/Ϫ brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate. Key words: aging; D-amino acids; DNA methylation; neurodegeneration; NMDA receptor Significance StatementThe enzyme D-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 of D-aspartate and keeping this D-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 that Ddo promoter demethylation enables the postnatal expression of Ddo. Moreover, persistent suppression of Ddo expression leads to persistent spillover of extracellular D-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.

Modeling DNA methylation by analyzing the individual configurations of single molecules

“…We used DDO as a model gene because, during development, this gene undergoes methylation in lung and gut (this manuscript) and demethylation in brain. 23 Moreover, the chosen system allowed us to study these processes in a natural context (tissues rather that cell lines), and to follow methylation changes occurring during development, thus avoiding any artifacts due to the action of drugs. Finally, the analyzed DDO methylation/demethylation processes are relevant for gene function, and thus may be a consequence of a deterministic phenomenon, since they are accompanied by gene activation (brain) 23 or gene repression (gut and lung).…”

“…Since we recently determined that a specific DDO promoter region undergoes demethylation in the brain during development, 23 here we checked whether the same region exhibits the opposite behavior in other tissues. DNA methylation was assessed through a strategy based on the locus-specific amplification of bisulfite-treated genomic DNA.…”

D-Amino Acids in the Nervous and Endocrine Systems

“…Although D-aspartate is abundant in the brains of rodents and humans during development, levels of D-aspartate are extremely decreased at postnatal stages and this reduction remains throughout adulthood [16, 67, 68, 71, 87, 88]. In contrast, DDO activity and its mRNA are exceedingly low at postnatal stages and increase after birth in rodents [88, 89]. Moreover, it has been shown that D-aspartate levels were significantly elevated in the CNS of Ddo knockout mice [88, 90–93].…”

“…In contrast, DDO activity and its mRNA are exceedingly low at postnatal stages and increase after birth in rodents [88, 89]. Moreover, it has been shown that D-aspartate levels were significantly elevated in the CNS of Ddo knockout mice [88, 90–93]. In addition, D-aspartate is found exclusively in the neurons of rats [67].…”

“…Therefore, a significant increase in levels of D-aspartate, enhancement of LTP, and increase in the dendritic length and spine density of neurons in adult Ddo knockout mice indicate that D-aspartate might also be generated and function in CNS during adulthood. However, persistent elevation in levels of D-aspartate in adult Ddo knockout mice has neurotoxic effects, such as activation of caspase-3 and apoptosis in neurons [88]. The expression of Ddo mRNA in postmortem brains of patients with schizophrenia was significantly higher than that in control individuals [95].…”