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.

Molecular and morphological findings in a sample of oral surgery patients: What can we learn for multivariate concepts for age estimation?

It has already been proposed that a combined use of different molecular and morphological markers of aging in multivariate models may result in a greater accuracy of age estimation. However, such an approach can be complex and expensive, and not every combination may be useful. The significance and usefulness of combined analyses of D-aspartic acid in dentine, pentosidine in dentine, DNA methylation in buccal swabs at five genomic regions (PDE4C, RPA2, ELOVL2, DDO, and EDARADD), and third molar mineralization were tested by investigating a sample of 90 oral surgery patients. Machine learning models for age estimation were trained and evaluated, and the contribution of each parameter to multivariate models was tested by assessment of the predictor importance. For models based on D-aspartic acid, pentosidine, and the combination of both, mean absolute errors (MAEs) of 2.93, 3.41, and 2.68 years were calculated, respectively. The additional inclusion of the five DNAm markers did not improve the results. The sole DNAm-based model revealed a MAE of 4.14 years. In individuals under 28 years of age, the combination of the DNAm markers with the third molar mineralization stages reduced the MAE from 3.85 to 2.81 years. Our findings confirm that the combination of parameters in multivariate models may be very useful for age estimation. However, the inclusion of many parameters does not necessarily lead to better results. It is a task for future research to identify the best selection of parameters for the different requirements in forensic practice.

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.

New Evidence on the Role of D-Aspartate Metabolism in Regulating Brain and Endocrine System Physiology: From Preclinical Observations to Clinical Applications.

The endogenous amino acids serine and aspartate occur at high concentrations in free D-form in mammalian organs, including the central nervous system and endocrine glands. D-serine (D-Ser) is largely localized in the forebrain structures throughout pre and postnatal life. Pharmacologically, D-Ser plays a functional role by acting as an endogenous coagonist at N-methyl-D-aspartate receptors (NMDARs). Less is known about the role of free D-aspartate (D-Asp) in mammals. Notably, D-Asp has a specific temporal pattern of occurrence. In fact, free D-Asp is abundant during prenatal life and decreases greatly after birth in concomitance with the postnatal onset of D-Asp oxidase expression, which is the only enzyme known to control endogenous levels of this molecule. Conversely, in the endocrine system, D-Asp concentrations enhance after birth during its functional development, thereby suggesting an involvement of the amino acid in the regulation of hormone biosynthesis. The substantial binding affinity for the NMDAR glutamate site has led us to investigate the in vivo implications of D-Asp on NMDAR-mediated responses. Herein we review the physiological function of free D-Asp and of its metabolizing enzyme in regulating the functions of the brain and of the neuroendocrine system based on recent genetic and pharmacological human and animal studies.

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 mouse d-aspartate oxidase.

D-amino acids research field has recently gained an increased interest since these atypical molecules have been discovered to play a plethora of different roles. In the mammalian central nervous system, d-aspartate (D-Asp) is critically involved in the regulation of glutamatergic neurotransmission by acting as an agonist of NMDA receptor. Accordingly, alterations in its metabolism have been related to different pathologies. D-Asp shows a peculiar temporal pattern of emergence during ontogenesis and soon after birth its brain levels are strictly regulated by the catabolic enzyme d-aspartate oxidase (DASPO), a FAD-dependent oxidase. Rodents have been widely used as in vivo models for deciphering molecular mechanisms and for testing novel therapeutic targets and drugs, but human targets can significantly differ. Based on these considerations, here we investigated the structural and functional properties of the mouse DASPO, in particular kinetic properties, ligand and flavin binding, oligomerization state and protein stability. We compared the obtained findings with those of the human enzyme (80% sequence identity) highlighting a different oligomeric state and a lower activity for the mouse DASPO, which apoprotein species exists in solution in two forms differing in FAD affinity. The features that distinguish mouse and human DASPO suggest that this flavoenzyme might control in a distinct way the brain D-Asp levels in different organisms.

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.

Structure and kinetic properties of human d-aspartate oxidase, the enzyme-controlling d-aspartate levels in brain.

d-Amino acids are the "wrong" enantiomers of amino acids as they are not used in proteins synthesis but evolved in selected functions. On this side, d-aspartate (d-Asp) plays several significant roles in mammals, especially as an agonist of N-methyl-d-aspartate receptors (NMDAR), and is involved in relevant diseases, such as schizophrenia and Alzheimer's disease. In vivo modulation of d-Asp levels represents an intriguing task to cope with such pathological states. As little is known about d-Asp synthesis, the only option for modulating the levels is via degradation, which is due to the flavoenzyme d-aspartate oxidase (DASPO). Here we present the first three-dimensional structure of a DASPO enzyme (from human) which belongs to the d-amino acid oxidase family. Notably, human DASPO differs from human d-amino acid oxidase (attributed to d-serine degradation, the main coagonist of NMDAR) showing peculiar structural features (a specific active site charge distribution), oligomeric state and kinetic mechanism, and a higher FAD affinity and activity. These results provide useful insights into the structure-function relationships of human DASPO: modulating its activity represents now a feasible novel therapeutic target.

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.

Rat d-aspartate oxidase is more similar to the human enzyme than the mouse enzyme.

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, as well as chronic pain. Thus, appropriate regulation of d-aspartate is believed to be important for maintaining proper neural activity in the nervous system. Accordingly, much attention has been paid to the role(s) of DDO in the metabolism of d-aspartate in vivo, and the physiological functions of DDO have been actively investigated using experimental rats and mice. However, detailed characterisation of rat DDO has not yet been performed, and little is known about species-specific differences in the properties of mammalian DDOs. In this study, the structural and enzymatic properties of purified recombinant rat, mouse and human DDOs were examined and compared. The results showed that rat DDO is more similar to human DDO than to mouse DDO. This work provides useful insight into the use of rats as an experimental model for investigating the biological significance of human DDO and/or d-aspartate. This article is part of a Special Issue entitled: d-Amino acids: biology in the mirror, edited by Dr. Loredano Pollegioni, Dr. Jean-Pierre Mothet and Dr. Molla Gianluca.

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.

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.