On the regulation of human D-aspartate oxidase.

The human flavoenzyme D-aspartate oxidase (hDASPO) controls the level of D-aspartate in the brain, a molecule acting as an agonist of NMDA receptors and modulator of AMPA and mGlu5 receptors. hDASPO-induced D-aspartate degradation prevents age-dependent deterioration of brain functions and is related to psychiatric disorders such as schizophrenia and autism. Notwithstanding this crucial role, less is known about hDASPO regulation. Here, we report that hDASPO is nitrosylated in vitro, while no evidence of sulfhydration and phosphorylation is apparent: nitrosylation affects the activity of the human flavoenzyme to a limited extent. Furthermore, hDASPO interacts with the primate-specific protein pLG72 (a well-known negative chaperone of D-amino acid oxidase, the enzyme deputed to D-serine degradation in the human brain), yielding a ~114 kDa complex, with a micromolar dissociation constant, promoting the flavoenzyme inactivation. At the cellular level, pLG72 and hDASPO generate a cytosolic complex: the expression of pLG72 negatively affects the hDASPO level by reducing its half-life. We propose that pLG72 binding may represent a protective mechanism aimed at avoiding cytotoxicity due to H2 O2 produced by the hDASPO enzymatic degradation of D-aspartate, especially before the final targeting to peroxisomes.

D-aspartate oxidase gene duplication induces social recognition memory deficit in mice and intellectual disabilities in humans.

The D-aspartate oxidase (DDO) gene encodes the enzyme responsible for the catabolism of D-aspartate, an atypical amino acid enriched in the mammalian brain and acting as an endogenous NMDA receptor agonist. Considering the key role of NMDA receptors in neurodevelopmental disorders, recent findings suggest a link between D-aspartate dysmetabolism and schizophrenia. To clarify the role of D-aspartate on brain development and functioning, we used a mouse model with constitutive Ddo overexpression and D-aspartate depletion. In these mice, we found reduced number of BrdU-positive dorsal pallium neurons during corticogenesis, and decreased cortical and striatal gray matter volume at adulthood. Brain abnormalities were associated with social recognition memory deficit at juvenile phase, suggesting that early D-aspartate occurrence influences neurodevelopmental related phenotypes. We corroborated this hypothesis by reporting the first clinical case of a young patient with severe intellectual disability, thought disorders and autism spectrum disorder symptomatology, harboring a duplication of a chromosome 6 region, including the entire DDO gene.

Glyoxylate reductase/hydroxypyruvate reductase regulates the free d-aspartate level in mammalian cells.

Multiple d-amino acids are present in mammalian cells, and these compounds have distinctive physiological functions. Among the free d-amino acids identified in mammals, d-aspartate plays critical roles in the neuroendocrine and endocrine systems, as well as in the central nervous system. Mammalian cells have the molecular apparatus necessary to take up, degrade, synthesize, and release d-aspartate. In particular, d-aspartate is degraded by d-aspartate oxidase (DDO), a peroxisome-localized enzyme that catalyzes the oxidative deamination of d-aspartate to generate oxaloacetate, hydrogen peroxide, and ammonia. However, little is known about the molecular mechanisms underlying d-aspartate homeostasis in cells. In this study, we established a cell line that overexpresses cytoplasm-localized DDO; this cell line cannot survive in the presence of high concentrations of d-aspartate, presumably because high levels of toxic hydrogen peroxide are produced by metabolism of abundant d-aspartate by DDO in the cytoplasm, where hydrogen peroxide cannot be removed due to the absence of catalase. Next, we transfected these cells with a complementary DNA library derived from the human brain and screened for clones that affected d-aspartate metabolism and improved cell survival, even when the cells were challenged with high concentrations of d-aspartate. The screen identified a clone of glyoxylate reductase/hydroxypyruvate reductase (GRHPR). Moreover, the GRHPR metabolites glyoxylate and hydroxypyruvate inhibited the enzymatic activity of DDO. Furthermore, we evaluated the effects of GRHPR and peroxisome-localized DDO on d- and l-aspartate levels in cultured mammalian cells. Our findings show that GRHPR contributes to the homeostasis of these amino acids in mammalian cells.

Cellular studies of the two main isoforms of human d-aspartate oxidase.

Human d-aspartate oxidase (hDASPO) is a FAD-dependent enzyme responsible for the degradation of d-aspartate (d-Asp). In the mammalian central nervous system, d-Asp behaves as a classical neurotransmitter, it is thought to be involved in neural development, brain morphology and behavior, and appears to be involved in several pathological states, such as schizophrenia and Alzheimer's disease. Apparently, the human DDO gene produces alternative transcripts encoding for three putative hDASPO isoforms, constituted by 341 (the 'canonical' form), 369, and 282 amino acids. Despite the increasing interest in hDASPO and its physiological role, little is known about these different isoforms. Here, the additional N-terminal peptide present in the hDASPO_369 isoform only has been identified in hippocampus of Alzheimer's disease female patients, while peptides corresponding to the remaining part of the protein were present in samples from male and female healthy controls and Alzheimer's disease patients. The hDASPO_369 isoform was largely expressed in E. coli as insoluble protein, hampering with its biochemical characterization. Furthermore, we generated U87 human glioblastoma cell clones stably expressing hDASPO_341 and, for the first time, hDASPO_369 isoforms; the latter protein showed a lower expression compared with the canonical isoform. Both protein isoforms are active (showing similar kinetic properties), localize to the peroxisomes, are very stable (a half-life of approximately 100 h has been estimated), and are primarily degraded through the ubiquitin-proteasome system. These studies shed light on the properties of hDASPO isoforms with the final aim to clarify the mechanisms controlling brain levels of the neuromodulator d-Asp.

Modeling DNA Methylation Profiles through a Dynamic Equilibrium between Methylation and Demethylation.

DNA methylation is a heritable epigenetic mark that plays a key role in regulating gene expression. Mathematical modeling has been extensively applied to unravel the regulatory mechanisms of this process. In this study, we aimed to investigate DNA methylation by performing a high-depth analysis of particular loci, and by subsequent modeling of the experimental results. In particular, we performed an in-deep DNA methylation profiling of two genomic loci surrounding the transcription start site of the D-Aspartate Oxidase and the D-Serine Oxidase genes in different samples (n = 51). We found evidence of cell-to-cell differences in DNA methylation status. However, these cell differences were maintained between different individuals, which indeed showed very similar DNA methylation profiles. Therefore, we hypothesized that the observed pattern of DNA methylation was the result of a dynamic balance between DNA methylation and demethylation, and that this balance was identical between individuals. We hence developed a simple mathematical model to test this hypothesis. Our model reliably captured the characteristics of the experimental data, suggesting that DNA methylation and demethylation work together in determining the methylation state of a locus. Furthermore, our model suggested that the methylation status of neighboring cytosines plays an important role in this balance.

New insights on the influence of free d-aspartate metabolism in the mammalian brain during prenatal and postnatal life.

Free d-aspartate is abundant in the mammalian embryonic brain. However, following the postnatal onset of the catabolic d-aspartate oxidase (DDO) activity, cerebral d-aspartate levels drastically decrease, remaining constantly low throughout life. d-Aspartate stimulates both glutamatergic NMDA receptors (NMDARs) and metabotropic Glu5 receptors. In rodents, short-term d-aspartate exposure increases spine density and synaptic plasticity, and improves cognition. Conversely, persistently high d-Asp levels produce NMDAR-dependent neurotoxic effects, leading to precocious neuroinflammation and cell death. These pieces of evidence highlight the dichotomous impact of d-aspartate signaling on NMDAR-dependent processes and, in turn, unveil a neuroprotective role for DDO in preventing the detrimental effects of excessive d-aspartate stimulation during aging. Here, we will focus on the in vivo influence of altered d-aspartate metabolism on the modulation of glutamatergic functions and its involvement in translational studies. Finally, preliminary data on the role of embryonic d-aspartate in the mouse brain will also be reviewed.

Biochemical characterization of d-aspartate oxidase from Caenorhabditis elegans: its potential use in the determination of free d-glutamate in biological samples.

d-Aspartate oxidase (DDO) is a flavin adenine dinucleotide (FAD)-containing flavoprotein that stereospecifically acts on acidic d-amino acids (i.e., free d-aspartate and d-glutamate). Mammalian DDO, which exhibits higher activity toward d-aspartate than d-glutamate, is presumed to regulate levels of d-aspartate in the body and is not thought to degrade d-glutamate in vivo. By contrast, three DDO isoforms are present in the nematode Caenorhabditis elegans, DDO-1, DDO-2, and DDO-3, all of which exhibit substantial activity toward d-glutamate as well as d-aspartate. In this study, we optimized the Escherichia coli culture conditions for production of recombinant C. elegans DDO-1, purified the protein, and showed that it is a flavoprotein with a noncovalently but tightly attached FAD. Furthermore, C. elegans DDO-1, but not mammalian (rat) DDO, efficiently and selectively degraded d-glutamate in addition to d-aspartate, even in the presence of various other amino acids. Thus, C. elegans DDO-1 might be a useful tool for determining these acidic d-amino acids in biological samples.

Prenatal expression of D-aspartate oxidase causes early cerebral D-aspartate depletion and influences brain morphology and cognitive functions at adulthood.

The free D-amino acid, D-aspartate, is abundant in the embryonic brain but significantly decreases after birth. Besides its intracellular occurrence, D-aspartate is also present at extracellular level and acts as an endogenous agonist for NMDA and mGlu5 receptors. These findings suggest that D-aspartate is a candidate signaling molecule involved in neural development, influencing brain morphology and behaviors at adulthood. To address this issue, we generated a knockin mouse model in which the enzyme regulating D-aspartate catabolism, D-aspartate oxidase (DDO), is expressed starting from the zygotic stage, to enable the removal of D-aspartate in prenatal and postnatal life. In line with our strategy, we found a severe depletion of cerebral D-aspartate levels (up to 95%), since the early stages of mouse prenatal life. Despite the loss of D-aspartate content, Ddo knockin mice are viable, fertile, and show normal gross brain morphology at adulthood. Interestingly, early D-aspartate depletion is associated with a selective increase in the number of parvalbumin-positive interneurons in the prefrontal cortex and also with improved memory performance in Ddo knockin mice. In conclusion, the present data indicate for the first time a biological significance of precocious D-aspartate in regulating mouse brain formation and function at adulthood.

D-Aspartate oxidase: distribution, functions, properties, and biotechnological applications.

Recently, substantial levels of acidic D-amino acids, such as D-aspartate and D-glutamate, have been identified in many organisms, from bacteria to mammals, suggesting that acidic D-amino acids have multiple physiological significances. Although acidic D-amino acids found in animals primarily originate from foodstuffs and/or bacteria, the D-aspartate-synthesizing enzyme aspartate racemase is identified in various animals. In eukaryotic organisms, acidic D-amino acids are primarily degraded by the flavoenzyme D-aspartate oxidase (DDO). DDO is found in multiple eukaryotic organisms and may play important roles in acidic D-amino acid utilization, elimination, and intracellular level regulation. Moreover, owing to its perfect enantioselectivity and stereoselectivity, DDO may be a valuable tool in several biotechnological applications, including the identification and quantification of acidic D-amino acids. In this mini-review, previous DDO reports are summarized and the potential bioengineering and biotechnological applications of DDO are discussed. Key Points ・Occurrence and distribution ofd-aspartate oxidase. ・Fundamental properties of d -aspartate oxidase of various eukaryotic organisms. ・Biotechnological applications and potential engineering ofd-aspartate oxidase.

Characterization and improvement of substrate-binding affinity of D-aspartate oxidase of the thermophilic fungus Thermomyces dupontii.

D-Aspartate oxidase (DDO) is a valuable enzyme that can be utilized in the determination of acidic D-amino acids and the optical resolution of a racemic mixture of acidic amino acids, which require its higher stability, higher catalytic activity, and higher substrate-binding affinity. In the present study, we identified DDO gene (TdDDO) of a thermophilic fungus, Thermomyces dupontii, and characterized the recombinant enzyme expressed in Escherichia coli. In addition, we generated a variant that has a higher substrate-binding affinity. The recombinant TdDDO expressed in E. coli exhibited oxidase activity toward acidic D-amino acids and a neutral D-amino acid, D-Gln, with the highest activity toward D-Glu. The Km and kcat values for D-Glu were 2.16 mM and 217 s-1, respectively. The enzyme had an optimum pH and temperature 8.0 and 60 °C, respectively, and was stable between pH 5.0 and 10.0, with a T50 of ca. 51 °C, which was much higher than that in DDOs from other origins. Enzyme stability decreased following a decrease in protein concentration, and externally added FAD could not repress the destabilization. The mutation of Phe248, potentially located in the active site of TdDDO, to Tyr residue, conserved in DDOs and D-amino acid oxidases, markedly increased substrate-binding affinity. The results showed the great potential of TdDDO and the variant for practical applications.

Free d-aspartate triggers NMDA receptor-dependent cell death in primary cortical neurons and perturbs JNK activation, Tau phosphorylation, and protein SUMOylation in the cerebral cortex of mice lacking d-aspartate oxidase activity.

In mammals, free d-aspartate (D-Asp) is abundant in the embryonic brain, while levels remain very low during adulthood as a result of the postnatal expression and activity of the catabolizing enzyme d-aspartate oxidase (DDO). Previous studies have shown that long-lasting exposure to nonphysiological, higher D-Asp concentrations in Ddo knockout (Ddo-/-) mice elicits a precocious decay of synaptic plasticity and cognitive functions, along with a dramatic age-dependent expression of active caspase 3, associated with increased cell death in different brain regions, including hippocampus, prefrontal cortex, and substantia nigra pars compacta. Here, we investigate the yet unclear molecular and cellular events associated with the exposure of abnormally high D-Asp concentrations in cortical primary neurons and in the brain of Ddo-/- mice. For the first time, our in vitro findings document that D-Asp induces in a time-, dose-, and NMDA receptor-dependent manner alterations in JNK and Tau phosphorylation levels, associated with pronounced cell death in primary cortical neurons. Moreover, observations obtained in Ddo-/- animals confirmed that high in vivo levels of D-Asp altered cortical JNK signaling, Tau phosphorylation and enhanced protein SUMOylation, indicating a robust indirect role of DDO activity in regulating these biochemical NMDA receptor-related processes. Finally, no gross modifications in D-Asp concentrations and DDO mRNA expression were detected in the cortex of patients with Alzheimer's disease when compared to age-matched healthy controls.

Secreted d-aspartate oxidase functions in C. elegans reproduction and development.

d-Aspartate oxidase (DDO) is a degradative enzyme that acts stereospecifically on free acidic D-amino acids such as d-aspartate and d-glutamate. d-Aspartate plays an important role in regulating neurotransmission, developmental processes, hormone secretion, and reproductive functions in mammals. In contrast, the physiological role of d-glutamate in mammals remains unclear. In Caenorhabditis elegans, the enzyme responsible for in vivo metabolism of d-glutamate is DDO-3, one of the three DDO isoforms, which is also required for normal self-fertility, hatching, and lifespan. In general, eukaryotic DDOs localize to subcellular peroxisomes in a peroxisomal targeting signal type 1 (PTS1)-dependent manner. However, DDO-3 does not contain a PTS1, but instead has a putative N-terminal signal peptide (SP). In this study, we found that DDO-3 is a secreted DDO, the first such enzyme to be described in eukaryotes. In hermaphrodites, DDO-3 was secreted from the proximal gonadal sheath cells in a SP-dependent manner and transferred to the oocyte surface. In males, DDO-3 was secreted from the seminal vesicle into the seminal fluid in a SP-dependent manner during mating with hermaphrodites. In both sexes, DDO-3 was secreted from the cells where it was produced into the body fluid and taken up by scavenger coelomocytes. Full-length DDO-3 transgene rescued all phenotypes elicited by the deletion of ddo-3, whereas a DDO-3 transgene lacking the putative SP did not. Together, these results indicate that secretion of DDO-3 is essential for its physiological functions.

DNA methylation landscape of the genes regulating D-serine and D-aspartate metabolism in post-mortem brain from controls and subjects with schizophrenia.

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.

Atopic asthma after rhinovirus-induced wheezing is associated with DNA methylation change in the SMAD3 gene promoter.

Children with rhinovirus-induced severe early wheezing have an increased risk of developing asthma later in life. The exact molecular mechanisms for this association are still mostly unknown. To identify potential changes in the transcriptional and epigenetic regulation in rhinovirus-associated atopic or nonatopic asthma, we analyzed a cohort of 5-year-old children (n = 45) according to the virus etiology of the first severe wheezing episode at the mean age of 13 months and to 5-year asthma outcome. The development of atopic asthma in children with early rhinovirus-induced wheezing was associated with DNA methylation changes at several genomic sites in chromosomal regions previously linked to asthma. The strongest changes in atopic asthma were detected in the promoter region of SMAD3 gene at chr 15q22.33 and introns of DDO/METTL24 genes at 6q21. These changes were validated to be present also at the average age of 8 years.

Structure-function relationships in human d-aspartate oxidase: characterisation of variants corresponding to known single nucleotide polymorphisms.

d-Aspartate oxidase (DDO) is a degradative enzyme that is stereospecific for the acidic amino acid d-aspartate, an endogenous agonist of the N-methyl-d-aspartate (NMDA) receptor. Dysregulation of NMDA receptor-mediated neurotransmission has been implicated in the onset of various neuropsychiatric disorders including schizophrenia and in chronic pain. Thus, appropriate regulation of the amount of d-aspartate is believed to be important for maintaining proper neural activity in the nervous system. Herein, the effects of the non-synonymous single nucleotide polymorphisms (SNPs) R216Q and S308N on several properties of human DDO were examined. Analysis of the purified recombinant enzyme showed that the R216Q and S308N substitutions reduce enzyme activity towards acidic d-amino acids, decrease the binding affinity for the coenzyme flavin adenine dinucleotide and decrease the temperature stability. Consistent with these findings, further experiments using cultured mammalian cells revealed elevated d-aspartate in cultures of R216Q and S308N cells compared with cells expressing wild-type DDO. Furthermore, accumulation of several amino acids other than d-aspartate also differed between these cultures. Thus, expression of DDO genes carrying the R216Q or S308N SNP substitutions may increase the d-aspartate content in humans and alter homeostasis of several other amino acids. This work may aid in understanding the correlation between DDO activity and the risk of onset of NMDA receptor-related diseases.

Olanzapine, but not clozapine, increases glutamate release in the prefrontal cortex of freely moving mice by inhibiting D-aspartate oxidase activity.

D-aspartate levels in the brain are regulated by the catabolic enzyme D-aspartate oxidase (DDO). D-aspartate activates NMDA receptors, and influences brain connectivity and behaviors relevant to schizophrenia in animal models. In addition, recent evidence reported a significant reduction of D-aspartate levels in the post-mortem brain of schizophrenia-affected patients, associated to higher DDO activity. In the present work, microdialysis experiments in freely moving mice revealed that exogenously administered D-aspartate efficiently cross the blood brain barrier and stimulates L-glutamate efflux in the prefrontal cortex (PFC). Consistently, D-aspartate was able to evoke L-glutamate release in a preparation of cortical synaptosomes through presynaptic stimulation of NMDA, mGlu5 and AMPA/kainate receptors. In support of a potential therapeutic relevance of D-aspartate metabolism in schizophrenia, in vitro enzymatic assays revealed that the second-generation antipsychotic olanzapine, differently to clozapine, chlorpromazine, haloperidol, bupropion, fluoxetine and amitriptyline, inhibits the human DDO activity. In line with in vitro evidence, chronic systemic administration of olanzapine induces a significant extracellular release of D-aspartate and L-glutamate in the PFC of freely moving mice, which is suppressed in Ddo knockout animals. These results suggest that the second-generation antipsychotic olanzapine, through the inhibition of DDO activity, increases L-glutamate release in the PFC of treated mice.

Methylome-wide association study of whole blood DNA in the Norfolk Island isolate identifies robust loci associated with age.

Epigenetic regulation of various genomic functions, including gene expression, provide mechanisms whereby an organism can dynamically respond to changes in its environment and modify gene expression accordingly. One epigenetic mechanism implicated in human aging and age-related disorders is DNA methylation. Isolated populations such as Norfolk Island (NI) should be advantageous for the identification of epigenetic factors related to aging due to reduced genetic and environmental variation. Here we conducted a methylome-wide association study of age using whole blood DNA in 24 healthy female individuals from the NI genetic isolate (aged 24-47 years). We analysed 450K methylation array data using a machine learning approach (GLMnet) to identify age-associated CpGs. We identified 497 CpG sites, mapping to 422 genes, associated with age, with 11 sites previously associated with age. The strongest associations identified were for a single CpG site in MYOF and an extended region within the promoter of DDO. These hits were validated in curated public data from 2316 blood samples (MARMAL-AID). This study is the first to report robust age associations for MYOF and DDO, both of which have plausible functional roles in aging. This study also illustrates the value of genetic isolates to reveal new associations with epigenome-level data.

Tracking the evolution of epialleles during neural differentiation and brain development: D-Aspartate oxidase as a model gene.

We performed ultra-deep methylation analysis at single molecule level of the promoter region of developmentally regulated D-Aspartate oxidase (Ddo), as a model gene, during brain development and embryonic stem cell neural differentiation. Single molecule methylation analysis enabled us to establish the effective epiallele composition within mixed or pure brain cell populations. In this framework, an epiallele is defined as a specific combination of methylated CpG within Ddo locus and can represent the epigenetic haplotype revealing a cell-to-cell methylation heterogeneity. Using this approach, we found a high degree of polymorphism of methylated alleles (epipolymorphism) evolving in a remarkably conserved fashion during brain development. The different sets of epialleles mark stage, brain areas, and cell type and unravel the possible role of specific CpGs in favoring or inhibiting local methylation. Undifferentiated embryonic stem cells showed non-organized distribution of epialleles that apparently originated by stochastic methylation events on individual CpGs. Upon neural differentiation, despite detecting no changes in average methylation, we observed that the epiallele distribution was profoundly different, gradually shifting toward organized patterns specific to the glial or neuronal cell types. Our findings provide a deep view of gene methylation heterogeneity in brain cell populations promising to furnish innovative ways to unravel mechanisms underlying methylation patterns generation and alteration in brain diseases.

ampliMethProfiler: a pipeline for the analysis of CpG methylation profiles of targeted deep bisulfite sequenced amplicons.

BACKGROUND: CpG sites in an individual molecule may exist in a binary state (methylated or unmethylated) and each individual DNA molecule, containing a certain number of CpGs, is a combination of these states defining an epihaplotype. Classic quantification based approaches to study DNA methylation are intrinsically unable to fully represent the complexity of the underlying methylation substrate. Epihaplotype based approaches, on the other hand, allow methylation profiles of cell populations to be studied at the single molecule level. For such investigations, next-generation sequencing techniques can be used, both for quantitative and for epihaplotype analysis. Currently available tools for methylation analysis lack output formats that explicitly report CpG methylation profiles at the single molecule level and that have suited statistical tools for their interpretation. RESULTS: Here we present ampliMethProfiler, a python-based pipeline for the extraction and statistical epihaplotype analysis of amplicons from targeted deep bisulfite sequencing of multiple DNA regions. CONCLUSIONS: ampliMethProfiler tool provides an easy and user friendly way to extract and analyze the epihaplotype composition of reads from targeted bisulfite sequencing experiments. ampliMethProfiler is written in python language and requires a local installation of BLAST and (optionally) QIIME tools. It can be run on Linux and OS X platforms. The software is open source and freely available at http://amplimethprofiler.sourceforge.net .

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 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(-/-)), 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 nonphysiological extracellular levels of free D-aspartate.