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.

It is long acknowledged that the N-methyl d-aspartate receptor co-agonist, d-serine, plays a crucial role in several N-methyl d-aspartate receptor-mediated physiological and pathological processes, including schizophrenia. Besides d-serine, another free d-amino acid, d-aspartate, is involved in the activation of N-methyl d-aspartate receptors acting as an agonist of this receptor subclass, and is abundantly detected in the developing human brain. Based on the hypothesis of N-methyl d-aspartate receptor hypofunction in the pathophysiology of schizophrenia and considering the ability of d-aspartate and d-serine to stimulate N-methyl d-aspartate receptor-dependent transmission, in the present work we assessed the concentration of these two d-amino acids in the post-mortem dorsolateral prefrontal cortex and hippocampus of patients with schizophrenia and healthy subjects. Moreover, in this cohort of post-mortem brain samples we investigated the spatiotemporal variations of d-aspartate and d-serine. Consistent with previous work, we found that d-aspartate content was selectively decreased by around 30% in the dorsolateral prefrontal cortex, but not in the hippocampus, of schizophrenia-affected patients, compared to healthy subjects. Interestingly, such selective reduction was associated to greater (around 25%) cortical activity of the enzyme responsible for d-aspartate catabolism, d-aspartate oxidase. Conversely, no significant changes were found in the methylation state and transcription of DDO gene in patients with schizophrenia, compared to control individuals, as well as in the expression levels of serine racemase, the major enzyme responsible for d-serine biosynthesis, which also catalyzes aspartate racemization. These results reveal the potential involvement of altered d-aspartate metabolism in the dorsolateral prefrontal cortex as a factor contributing to dysfunctional N-methyl d-aspartate receptor-mediated transmission in schizophrenia.

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The spatio-temporal regulation of genes involved in the synthesis and degradation of D-serine and D-aspartate such as serine racemase (SR), D-amino acid oxidase (DAO), G72 and D-aspartate oxidase (DDO), play pivotal roles in determining the correct levels of these D-amino acids in the human brain. Here we provide a comprehensive analysis of mRNA expression and DNA methylation status of these genes in post-mortem samples from hippocampus, dorsolateral prefrontal cortex, and cerebellum from patients with schizophrenia and non-psychiatric controls. DNA methylation analysis was performed at an ultradeep level, measuring individual epialleles frequency by single molecule approach. Differential CpG methylation and expression was detected across different brain regions, although no significant correlations were found with diagnosis. G72 showed the highest CpG and non-CpG methylation degree, which may explain the repression of G72 transcription in the brain regions considered here. Conversely, in line with the sustained SR mRNA expression in the analyzed areas, very low methylation levels were detected at this gene’s regulatory regions. Furthermore, for DAO and DDO, our single-molecule methylation approach demonstrated that analysis of epiallele distribution was able to detect differences in DNA methylation representing area-specific methylation signatures, which are likely not detectable with targeted or genome-wide classic methylation analyses.

BackgroundProgrammed epigenetic modifications occurring at early postnatal brain developmental stages may have a long-lasting impact on brain function and complex behavior throughout life. Notably, it is now emerging that several genes that undergo perinatal changes in DNA methylation are associated with neuropsychiatric disorders. In this context, we envisaged that epigenetic modifications during the perinatal period may potentially drive essential changes in the genes regulating brain levels of critical neuromodulators such as d-serine and d-aspartate. Dysfunction of this fine regulation may contribute to the genesis of schizophrenia or other mental disorders, in which altered levels of d-amino acids are found. We recently demonstrated that Ddo, the d-aspartate degradation gene, is actively demethylated to ultimately reduce d-aspartate levels. However, the role of epigenetics as a mechanism driving the regulation of appropriate d-ser levels during brain development has been poorly investigated to date.MethodsWe performed comprehensive ultradeep DNA methylation and hydroxymethylation profiling along with mRNA expression and HPLC-based d-amino acids level analyses of genes controlling the mammalian brain levels of d-serine and d-aspartate. DNA methylation changes occurring in specific cerebellar cell types were also investigated. We conducted high coverage targeted bisulfite sequencing by next-generation sequencing and single-molecule bioinformatic analysis.ResultsWe report consistent spatiotemporal modifications occurring at the Dao gene during neonatal development in a specific brain region (the cerebellum) and within specific cell types (astrocytes) for the first time. Dynamic demethylation at two specific CpG sites located just downstream of the transcription start site was sufficient to strongly activate the Dao gene, ultimately promoting the complete physiological degradation of cerebellar d-serine a few days after mouse birth. High amount of 5′-hydroxymethylcytosine, exclusively detected at relevant CpG sites, strongly evoked the occurrence of an active demethylation process.ConclusionThe present investigation demonstrates that robust and selective demethylation of two CpG sites is associated with postnatal activation of the Dao gene and consequent removal of d-serine within the mouse cerebellum. A single-molecule methylation approach applied at the Dao locus promises to identify different cell-type compositions and functions in different brain areas and developmental stages.Electronic supplementary materialThe online version of this article (10.1186/s13148-019-0732-z) contains supplementary material, which is available to authorized users.