D-aspartate Oxidase Research Articles

Screening of Crucial Cytosolicproteins Interconnecting the Endoplasmic Reticulum and Mitochondria in Parkinson's Disease and the Impact of Anti-Parkinson Drugs in the Preservation of Organelle Connectivity.

Mitochondrial dysfunction is well-established in Parkinson's disease (PD); however, its dysfunctions associating with cell organelle connectivity remain unknown. We aimed to establish the crucial cytosolic protein involved in organelle connectivity between mitochondria and the endopalmic reticulum (ER) through a computational approach by constructing an organelle protein network to extract functional clusters presenting the crucial PD protein connecting organelles. Then, we assessed the influence of anti-parkinsonism drugs (n = 35) on the crucial protein through molecular docking and molecular dynamic simulation and further validated its gene expression in PD participants under, istradefylline (n = 25) and amantadine (n = 25) treatment. Based on our investigation, D-aspartate oxidase (DDO )protein was found to be the critical that connects both mitochondria and the ER. Further, molecular docking showed that istradefylline has a high affinity (-9.073 kcal/mol) against DDO protein, which may disrupt mitochondrial-ER connectivity. While amantadine (-4.53 kcal/mol) shows negligible effects against DDO that contribute to conformational changes in drug binding, Successively, DDO gene expression was downregulated in istradefylline-treated PD participants, which elucidated the likelihood of an istradefylline off-target mechanism. Overall, our findings illuminate the off-target effects of anti-parkinsonism medications on DDO protein, enabling the recommendation of off-target-free PD treatments.

3-Chloropropane-1,2-diol exposure adversely influenced the bio-accessibility signatures of digested infant foods by suppressing the destabilization of α-lactalbumin and d-aspartate oxidase in a dose-dependent manner.

The potential mechanisms about the health risks of endogenous 3-MCPD remain elusive. Here, we researched the influences of 3-MCPD on the metabolic landscape of digested goat infant formulas via integrative UHPLC-Q-Orbitrap HRMS-MS/MS-based peptidomics and metabolomics (%RSDs≤7.35%, LOQ 2.99-58.77μgkg-1). Digested goat infant formulas under 3-MCPD-interference caused metabolic perturbation by down-regulating levels of peptides VGINYWLAHK (5.98-0.72mgkg-1) and HLMCLSWQ (3.25-0.72mgkg-1) pertained to health-promoting bioactive components, and accelerated the down-regulation of non-essential amino acids (AAs, l-tyrosine 0.88-0.39mgkg-1, glutamic acid 8.83-0.88μgkg-1, and d-aspartic acid 2.93-0.43μgkg-1), semi-essential AA (l-arginine 13.06-8.12μgkg-1) and essential AAs (l-phenylalanine 0.49-0.05mgkg-1) that provide nutritional value. Peptidomics and metabolomics interactions elucidated that 3-MCPD altered the stability of α-lactalbumin and d-aspartate oxidase in a dose-dependent manner, and affected the flavor perception of goat infant formulas, leading to a decline of nutritional value of goat infant formulas.

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 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 to a limited extent the activity of the human flavoenzyme. 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. This article is protected by copyright. All rights reserved.

Improvement of Learning and Memory by Elevating Brain D-Aspartate in a Mouse Model of Fragile X Syndrome.

Fragile X syndrome (FXS) is an inherited human mental retardation that arises from expansion of a CGG repeat in the Fmr1 gene, causing loss of the fragile X mental retardation protein (FMRP). It is reported that N-methyl-D-aspartate receptor (NMDAR)-mediated facilitation of long-term potentiation (LTP) and fear memory are impaired in Fmr1 knockout (KO) mice. In this study, biological, pharmacological, and electrophysiological techniques were performed to determine the roles of D-aspartate (D-Asp), a modulator of NMDAR, and its metabolizing enzyme D-aspartate oxidase (DDO) in Fmr1 KO mice. Levels of D-Asp were decreased in the medial prefrontal cortex (mPFC ); however, the levels of its metabolizing enzyme DDO were increased. Electrophysiological recordings indicated that oral drinking of D-Asp recovered LTP induction in mPFC from Fmr1 KO mice. Moreover, chronic oral administration of D-Asp reversed behavioral deficits of cognition and locomotor coordination in Fmr1 KO mice. The therapeutic action of D-Asp was partially through regulating functions of NMDARs and mGluR5/mTOR/4E-BP signaling pathways. In conclusion, supplement of D-Asp may benefit for synaptic plasticity and behaviors in Fmr1 KO mice and offer a potential therapeutic strategy for FXS.

PHYSIOLOGICAL ROLE OF D-AMINO ACIDS AND BIOANALYTICAL POTENTIAL OF D-AMINO ACID OXIDASES

D-amino acid oxidase (DAAO) plays an important role in the functioning of both prokaryotes and eukaryotes. DAAO is increasingly being used in practice, including for the determination of D-amino acids in complex samples, including human tissues and fl uids. There are generally two types of DAAO in all organisms. The fi rst type is an enzyme highly specifi c for D-aspartate and has its own name D-aspartate oxidase (DASPO). DAAO of the second type is characterized by a wide spectrum of substrate specificity, with preference for one or another D-amino acid varying from source to source. The activity of DAAO with a large number of substrates greatly complicates the selective determination of a particular D-amino acid. The problem is often solved by choosing an enzyme that, under the conditions of analysis, has low or no activity with other D-amino acids present in the sample. For the convenience of selecting a particular enzyme, we have collected and analyzed literature data on the catalytic parameters of known DAAOs with the most important D-amino acids. In addition, similar data are presented for novel recombinant DAAOs from the methylotrophic yeast Ogataea parapolymorpha DL-1. Analysis of the data shows that, with the D-amino acid series, the new OpaDASPO and OpaDAAO have the highest catalytic parameters.

Development of an aminotransferase-driven biocatalytic cascade for deracemization of d,l-phosphinothricin.

2-oxo-4-[(hydroxy)(methyl)phosphinoyl]butyric acid (PPO) is the essential precursor keto acid for the asymmetric biosynthesis of herbicide l-phosphinothricin (l-PPT). Developing a biocatalytic cascade for PPO production with high efficiency and low cost is highly desired. Herein, a d-amino acid aminotransferase from Bacillus sp. YM-1 (Ym DAAT) with high activity (48.95 U/mg) and affinity (Km = 27.49 mM) toward d-PPT was evaluated. To circumvent the inhibition of by-product d-glutamate (d-Glu), an amino acceptor (α-ketoglutarate) regeneration cascade was constructed as a recombinant Escherichia coli (E. coli D), by coupling Ym d-AAT, d-aspartate oxidase from Thermomyces dupontii (TdDDO) and catalase from Geobacillus sp. CHB1. Moreover, the regulation of the ribosome binding site was employed to overcome the limiting step of expression toxic protein TdDDO in E. coli BL21(DE3). The aminotransferase-driven whole-cell biocatalytic cascade (E. coli D) showed superior catalytic efficiency for the synthesis of PPO from d,l-phosphinothricin(d,l-PPT). It revealed the production of PPO exhibited high space-time yield (2.59 g L-1 h-1 ) with complete conversion of d-PPT to PPO at high substrate concentration (600 mM d,l-PPT) in 1.5 L reaction system. This study first provides the synthesis of PPO from d,l-PPT employing an aminotransferase-driven biocatalytic cascade.

D-Aspartate Depletion Perturbs Steroidogenesis and Spermatogenesis in Mice.

High levels of free D-aspartate (D-Asp) are present in vertebrate testis during post-natal development, coinciding with the onset of testosterone production, which suggests that this atypical amino acid might participate in the regulation of hormone biosynthesis. To elucidate the unknown role of D-Asp on testicular function, we investigated steroidogenesis and spermatogenesis in a one-month-old knockin mouse model with the constitutive depletion of D-Asp levels due to the targeted overexpression of D-aspartate oxidase (DDO), which catalyzes the deaminative oxidation of D-Asp to generate the corresponding α-keto acid, oxaloacetate, hydrogen peroxide, and ammonium ions. In the Ddo knockin mice, we found a dramatic reduction in testicular D-Asp levels, accompanied by a significant decrease in the serum testosterone levels and testicular 17β-HSD, the enzyme involved in testosterone biosynthesis. Additionally, in the testes of these Ddo knockin mice, the expression of PCNA and SYCP3 proteins decreased, suggesting alterations in spermatogenesis-related processes, as well as an increase in the cytosolic cytochrome c protein levels and TUNEL-positive cell number, which indicate an increase in apoptosis. To further investigate the histological and morphometric testicular alterations in Ddo knockin mice, we analyzed the expression and localization of prolyl endopeptidase (PREP) and disheveled-associated activator of morphogenesis 1 (DAAM1), two proteins involved in cytoskeletal organization. Our results showed that the testicular levels of DAAM1 and PREP in Ddo knockin mice were different from those in wild-type animals, suggesting that the deficiency of D-Asp is associated with overall cytoskeletal disorganization. Our findings confirmed that physiological D-Asp influences testosterone biosynthesis and plays a crucial role in germ cell proliferation and differentiation, which are required for successful reproduction.

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.

Rational and Translational Implications of D-Amino Acids for Treatment-Resistant Schizophrenia: From Neurobiology to the Clinics.

Schizophrenia has been conceptualized as a neurodevelopmental disorder with synaptic alterations and aberrant cortical–subcortical connections. Antipsychotics are the mainstay of schizophrenia treatment and nearly all share the common feature of dopamine D2 receptor occupancy, whereas glutamatergic abnormalities are not targeted by the presently available therapies. D-amino acids, acting as N-methyl-D-aspartate receptor (NMDAR) modulators, have emerged in the last few years as a potential augmentation strategy in those cases of schizophrenia that do not respond well to antipsychotics, a condition defined as treatment-resistant schizophrenia (TRS), affecting almost 30–40% of patients, and characterized by serious cognitive deficits and functional impairment. In the present systematic review, we address with a direct and reverse translational perspective the efficacy of D-amino acids, including D-serine, D-aspartate, and D-alanine, in poor responders. The impact of these molecules on the synaptic architecture is also considered in the light of dendritic spine changes reported in schizophrenia and antipsychotics’ effect on postsynaptic density proteins. Moreover, we describe compounds targeting D-amino acid oxidase and D-aspartate oxidase enzymes. Finally, other drugs acting at NMDAR and proxy of D-amino acids function, such as D-cycloserine, sarcosine, and glycine, are considered in the light of the clinical burden of TRS, together with other emerging molecules.

Free d-Amino Acids in Salivary Gland in Rat.

Simple SummaryParotid, submandibular, and sublingual glands in rat were found to contain high concentrations of d-aspartic acid and low ones of d-serine and d-alanine. In addition to d-amino acid oxidase and d-aspartate oxidase, serine racemase was also detected in all three of these major salivary glands, as were N-methyl-d-aspartic acid receptor subunits NR1 and NR2D, but not NR2A, NR2B, or NR2C.Free d-amino acids, which are enantiomers of l-amino acids, are found in mammals, including humans, and play an important role in a range of physiological functions in the central nervous system and peripheral tissues. Several d-amino acids have been observed in saliva, but their origin and the enzymes involved in their metabolism and catabolism remain to be clarified. In the present study, large amounts of d-aspartic acid and small amounts of d-serine and d-alanine were detected in all three major salivary glands in rat. No other d-enantiomers were detected. Protein expression of d-amino acid oxidase and d-aspartate oxidase, the enzymes responsible for the oxidative deamination of neutral and dicarboxylic d-amino acids, respectively, were detected in all three types of salivary gland. Furthermore, protein expression of the d-serine metabolic enzyme, serine racemase, in parotid glands amounted to approximately 40% of that observed in the cerebral cortex. The N-methyl-d-aspartic acid subunit proteins NR1 and NR2D were detected in all three major salivary glands. The results of the present study suggest that d-amino acids play a physiological role in a range of endocrine and exocrine function in salivary glands.

Regulation of d-Aspartate Oxidase Gene Expression by Pyruvate Metabolism in the Yeast Cryptococcus humicola.

d-Aspartate oxidase (DDO) is a peroxisomal flavoenzyme that catalyzes the oxidative deamination of acidic d-amino acids. In the yeast Cryptococcus humicola strain UJ1, the enzyme ChDDO is essential for d-Asp utilization and is expressed only in the presence of d-Asp. Pyruvate carboxylase (Pyc) catalyzes the conversion of pyruvate to oxaloacetate and is involved in the import and activation of certain peroxisomal flavoenzymes in yeasts. In this study, we analyzed the role of Pyc in the expression of ChDDO gene in C. humicola strain UJ1. PYC gene disruption (∆Chpyc1) in strain UJ1 resulted in growth retardation on glucose and NH4Cl medium. The growth was restored by supplying oxaloacetate from l-Asp or α-ketoglutarate by a transaminase. On the other hand, the supply of oxaloacetate from d-Asp by ChDDO was not able to prevent growth retardation because of a significant decrease in ChDDO gene expression at the transcriptional level. The addition of pyruvate significantly decreased ChDDO gene transcription in the ∆Chpyc1 strain but increased the same in the wild-type strain, even though the intracellular pyruvate content was similar in both strains. These results suggest that ChDDO gene expression might be regulated by pyruvate metabolism, as well as by the presence of d-Asp.

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.

Human D-aspartate Oxidase: A Key Player in D-aspartate Metabolism.

In recent years, the D-enantiomers of amino acids have been recognized as natural molecules present in all kingdoms, playing a variety of biological roles. In humans, d-serine and d-aspartate attracted attention for their presence in the central nervous system. Here, we focus on d-aspartate, which is involved in glutamatergic neurotransmission and the synthesis of various hormones. The biosynthesis of d-aspartate is still obscure, while its degradation is due to the peroxisomal flavin adenine dinucleotide (FAD)-containing enzyme d-aspartate oxidase. d-Aspartate emergence is strictly controlled: levels decrease in brain within the first days of life while increasing in endocrine glands postnatally and through adulthood. The human d-aspartate oxidase (hDASPO) belongs to the d-amino acid oxidase-like family: its tertiary structure closely resembles that of human d-amino acid oxidase (hDAAO), the enzyme that degrades neutral and basic d-amino acids. The structure-function relationships of the physiological isoform of hDASPO (named hDASPO_341) and the regulation of gene expression and distribution and properties of the longer isoform hDASPO_369 have all been recently elucidated. Beyond the substrate preference, hDASPO and hDAAO also differ in kinetic efficiency, FAD-binding affinity, pH profile, and oligomeric state. Such differences suggest that evolution diverged to create two different ways to modulate d-aspartate and d-serine levels in the human brain. Current knowledge about hDASPO is shedding light on the molecular mechanisms underlying the modulation of d-aspartate levels in human tissues and is pushing novel, targeted therapeutic strategies. Now, it has been proposed that dysfunction in NMDA receptor-mediated neurotransmission is caused by disrupted d-aspartate metabolism in the nervous system during the onset of various disorders (such as schizophrenia): the design of suitable hDASPO inhibitors aimed at increasing d-aspartate levels thus represents a novel and useful form of therapy.

Development of an enzymatic screening method for d-aspartate-producing lactic acid bacteria

d-Aspartate (d-Asp) is an important intermediate for synthetic penicillin and an endogenous amino acid that plays important roles in the endocrine and nervous systems in animals including humans. Lactic acid bacteria (LABs) have been used as probiotics in humans, and some LAB species produce d-Asp as a component of cell wall peptidoglycan. LAB strains with greater d-Asp production would therefore be valuable for industrial d-Asp production. In this study, we developed an enzymatic screening method for d-Asp-producing LABs and isolated a strain with high d-Asp production. The d-Asp concentration in the culture medium was colorimetrically estimated up to 4 mM using d-aspartate oxidase (ChDDO) from the yeast Cryptococcus humicola strain UJ1 coupled with horseradish peroxidase, although a more accurate determination required correction because of interference by the medium component Mn2+. We isolated 628 LAB strains from various foods and screened them for d-Asp production using the enzymatic d-Asp assay method. The screening identified 13 d-Asp-producing LAB strains, which were suggested to belong to the genera Latilactobacillus, Levilactobacillus, Lactococcus, and Enterococcus. d-Asp production ability was likely to widely differ among the strains in the same genera and species. One strain, named strain WDN19, produced much higher d-Asp levels (1.84 mM), and it was closely related to Latilactobacillus curvatus. These results indicated that the enzymatic screening method was useful for identifying and isolating d-Asp-producing LABs rapidly and easily, and it might provide novel findings regarding d-Asp production by LABs.

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 100h 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.

Transcriptome analysis reveals hepatopancreatic genes and pathways associated with metabolism responding to water salinities during the overwintering of mud crab Scylla paramamosain

Overwintering is an important part of the conservation of Scylla paramamosain, and salinity is a primary parameter affecting this species’ metabolism at this particular time. We compared and analysed gene sequences responsible for the metabolism of S. paramamosain at 4‰ versus 25‰ salinities using transcriptome analysis, in order to find out the optimum salinity for S. paramamosain during overwintering. A total of 5895 genes were detected, of which 105 down-regulated genes and 131 up-regulated genes were associated with metabolism (p < 0.05). Among these genes related to metabolic pathways (117 pathways), we identified D-aspartate oxidase, D-amino acid oxidase, creatine kinase, sarcosine oxidase, citrate synthase, transferase, glycogen synthase, hexokinase, carbonic anhydrase, glutamine synthetase, V-type H+-transporting ATPase, 3′-phosphoadenosine 5′-phosphosulphate synthase, fatty acid synthase and others. Additionally, the consistency between qRT-PCR results and transcriptome data reflected the reliability of transcriptome results in the present study. Compared with the 25‰ salinity group, the 4‰ salinity group promoted energy metabolism, amino acid metabolism, lipid metabolism and carbohydrate metabolism by regulating metabolism-related genes, which made the overwintering S. paramamosain more active in the low-salt environment, and hepatopancreas consumed more energy to maintain life activities. These results improve our understanding on molecular mechanisms and metabolic pathways associated with salinity changes in S. paramamosain during overwintering. The data presented in this study are also the first to reveal the molecular mechanisms of S. paramamosain during overwintering in response to different salinities at the gene level.

Identification of an Acidic Amino Acid Permease Involved in d-Aspartate Uptake in the Yeast Cryptococcus humicola.

d-aspartate oxidase (DDO) catalyzes the oxidative deamination of acidic d-amino acids, and its production is induced by d-Asp in several eukaryotes. The yeast Cryptococcus humicola strain UJ1 produces large amounts of DDO (ChDDO) only in the presence of d-Asp. In this study, we analyzed the relationship between d-Asp uptake by an amino acid permease (Aap) and the inducible expression of ChDDO. We identified two acidic Aap homologs, named “ChAap4 and ChAap5,” in the yeast genome sequence. ChAAP4 deletion resulted in partial growth defects on d-Asp as well as l-Asp, l-Glu, and l-Phe at pH 7, whereas ChAAP5 deletion caused partial growth defects on l-Phe and l-Lys, suggesting that ChAap4 might participate in d-Asp uptake as an acidic Aap. Interestingly, the growth of the Chaap4 strain on d- or l-Asp was completely abolished at pH 10, suggesting that ChAap4 is the only Aap responsible for d- and l-Asp uptake under high alkaline conditions. In addition, ChAAP4 deletion significantly decreased the induction of DDO activity and ChDDO transcription in the presence of d-Asp. This study revealed that d-Asp uptake by ChAap4 might be involved in the induction of ChDDO expression by d-Asp.

Prenatal and Early Postnatal Cerebral d-Aspartate Depletion Influences l-Amino Acid Pathways, Bioenergetic processes, and Developmental Brain Metabolism.

d-Amino acids were believed to occur only in bacteria and invertebrates. Today, it is well known that d-amino acids are also present in mammalian tissues in a considerable amount. In particular, high levels of free d-serine (d-Ser) and d-aspartate (d-Asp) are found in the brain. While the functions of d-Ser are well known, many questions remain unanswered regarding the role of d-Asp in the central nervous system. d-Asp is very abundant at the embryonic stage, while it strongly decreases after birth because of the expression of d-aspartate oxidase (Ddo) enzyme, which catalyzes the oxidation of this d-amino acid into oxaloacetate, ammonium, and hydrogen peroxide. Pharmacologically, d-Asp acts as an endogenous agonist of N-methyl d-aspartate and mGlu5 receptors, which are known to control fundamental brain processes, including brain development, synaptic plasticity, and cognition. In this work, we studied a recently generated knockin mouse model (R26ddo/ddo), which was designed to express DDO beginning at the zygotic stage. This strategy enables d-Asp to be almost eliminated in both prenatal and postnatal lives. To understand which biochemical pathways are affected by depletion of d-Asp, in this study, we carried out a metabolomic and lipidomic study of ddo knockin brains at different stages of embryonic and postnatal development, combining nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRMS) techniques. Our study shows that d-Asp deficiency in the brain influences amino acid pathways such as threonine, glycine, alanine, valine, and glutamate. Interestingly, d-Asp is also correlated with metabolites involved in brain development and functions such as choline, creatine, phosphocholine (PCho), glycerophosphocholine (GPCho), sphingolipids, and glycerophospholipids, as well as metabolites involved in brain energy metabolism, such as GPCho, glucose, and lactate.

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.

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.