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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., (© 2021 Federation of European Biochemical Societies.)

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-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., (© 2019 Federation of American Societies for Experimental Biology.)

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., (Copyright © 2019. Published by Elsevier Inc.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

We have investigated the relevance of d-aspartate oxidase, the only enzyme known to selectively degrade d-aspartate (d-Asp), in modulating glutamatergic system homeostasis. Interestingly, the lack of the Ddo gene, by raising d-Asp content, induces a substantial increase in extracellular glutamate (Glu) levels in Ddo-mutant brains. Consistent with an exaggerated and persistent N-methyl-d-aspartate receptor (NMDAR) stimulation, we documented in Ddo knockouts severe age-dependent structural and functional alterations mirrored by expression of active caspases 3 and 7 along with appearance of dystrophic microglia and reactive astrocytes. In addition, prolonged elevation of d-Asp triggered in mutants alterations of NMDAR-dependent synaptic plasticity associated to reduction of hippocampal GluN1 and GluN2B subunits selectively located at synaptic sites and to increase in the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-to-N-methyl-d-aspartate ratio. These effects, all of which converged on a progressive hyporesponsiveness at NMDAR sites, functionally resulted in a greater vulnerability to phencyclidine-induced prepulse inhibition deficits in mutants. In conclusion, our results indicate that d-aspartate oxidase, by strictly regulating d-Asp levels, impacts on the homeostasis of glutamatergic system, thus preventing accelerated neurodegenerative processes., (Copyright © 2015 Elsevier Inc. All rights reserved.)

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. [ABSTRACT FROM AUTHOR]

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 (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. [ABSTRACT FROM AUTHOR]

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.

Besides d-serine, another d-amino acid with endogenous occurrence in the mammalian brain, d-aspartate, has been recently shown to influence NMDA receptor (NMDAR)-mediated transmission. d-aspartate is present in the brain at extracellular level in nanomolar concentrations, binds to the agonist site of NMDARs and activates this subclass of glutamate receptors. Along with its direct effect on NMDARs, d-aspartate can also evoke considerable l-glutamate release in specific brain areas through the presynaptic activation of NMDA, AMPA/kainate and mGlu5 receptors. d-aspartate is enriched in the embryonic brain of rodents and humans and its concentration strongly decreases after birth, due to the post-natal expression of the catabolising enzyme d-aspartate oxidase (DDO). Based on the hypothesis of NMDAR hypofunction in schizophrenia pathogenesis, recent preclinical and clinical studies suggested a relationship between perturbation of d-aspartate metabolism and this psychiatric disorder. Consistently, neurophysiological and behavioral characterization of Ddo knockout (Ddo−/−) and d-aspartate-treated mice highlighted that abnormally higher endogenous d-aspartate levels significantly increase NMDAR-mediated synaptic plasticity, neuronal spine density and memory. Remarkably, increased d-aspartate levels influence schizophrenia-like phenotypes in rodents, as indicated by improved fronto-hippocampal connectivity, attenuated prepulse inhibition deficits and reduced activation of neuronal circuitry induced by phencyclidine exposure. In healthy humans, a genetic polymorphism associated with reduced prefrontal DDO gene expression predicts changes in prefrontal phenotypes including greater gray matter volume and enhanced functional activity during working memory. Moreover, neurochemical detections in post-mortem brain of schizophrenia-affected patients have shown significantly reduced d-aspartate content in prefrontal regions, associated with increased DDO mRNA expression or DDO enzymatic activity. Overall, these findings suggest a possible involvement of dysregulated embryonic d-aspartate metabolism in schizophrenia pathophysiology and, in turn, highlight the potential use of free d-aspartate supplementation as a new add-on therapy for treating the cognitive symptoms of this mental illness.

Besides d-serine, another d-amino acid with endogenous occurrence in the mammalian brain, d-aspartate, has been recently shown to influence NMDA receptor (NMDAR)-mediated transmission. d-aspartate is present in the brain at extracellular level in nanomolar concentrations, binds to the agonist site of NMDARs and activates this subclass of glutamate receptors. Along with its direct effect on NMDARs, d-aspartate can also evoke considerable l-glutamate release in specific brain areas through the presynaptic activation of NMDA, AMPA/kainate and mGlu5 receptors. d-aspartate is enriched in the embryonic brain of rodents and humans and its concentration strongly decreases after birth, due to the post-natal expression of the catabolising enzyme d-aspartate oxidase (DDO). Based on the hypothesis of NMDAR hypofunction in schizophrenia pathogenesis, recent preclinical and clinical studies suggested a relationship between perturbation of d-aspartate metabolism and this psychiatric disorder. Consistently, neurophysiological and behavioral characterization of Ddo knockout (Ddo −/−) and d-aspartate-treated mice highlighted that abnormally higher endogenous d-aspartate levels significantly increase NMDAR-mediated synaptic plasticity, neuronal spine density and memory. Remarkably, increased d-aspartate levels influence schizophrenia-like phenotypes in rodents, as indicated by improved fronto-hippocampal connectivity, attenuated prepulse inhibition deficits and reduced activation of neuronal circuitry induced by phencyclidine exposure. In healthy humans, a genetic polymorphism associated with reduced prefrontal DDO gene expression predicts changes in prefrontal phenotypes including greater gray matter volume and enhanced functional activity during working memory. Moreover, neurochemical detections in post-mortem brain of schizophrenia-affected patients have shown significantly reduced d-aspartate content in prefrontal regions, associated with increased DDO mRNA expression or DDO enzymatic activity. Overall, these findings suggest a possible involvement of dysregulated embryonic d-aspartate metabolism in schizophrenia pathophysiology and, in turn, highlight the potential use of free d-aspartate supplementation as a new add-on therapy for treating the cognitive symptoms of this mental illness. [ABSTRACT FROM AUTHOR]

Free d-aspartate ( d-Asp) occurs in substantial amounts in glandular tissues. This paper reviews the existing work on d-Asp in vertebrate exocrine and endocrine glands, with emphasis on functional roles. Endogenous d-Asp was detected in salivary glands. High d-Asp levels in the parotid gland during development suggest an involvement of the amino acid in the regulation of early developmental phases and/or differentiation processes. d-Asp has a prominent role in the Harderian gland, where it elicits exocrine secretion through activation of the ERK1/2 pathway. Interestingly, the increase in NOS activity associated with d-Asp administration in the Harderian gland suggests a potential capability of d-Asp to induce vasodilatation. In mammals, an increase in local concentrations of d-Asp facilitates the secretion of anterior pituitary hormones, i.e., PRL, LH and GH, whereas it inhibits the secretion of POMC/α-MSH from the intermediate pituitary and of oxytocin from the posterior pituitary. d-Asp also acts as a negative regulator for melatonin synthesis in the pineal gland. Further, d-Asp can stereo-specifically modulate the production of sex steroids, thus taking part in the endocrine control of reproductive activity. Although d-Asp receptors remain to be characterized, gene expression of NR1 and NR2 subunits of NMDAr responds to d-Asp in the testis. [ABSTRACT FROM AUTHOR]

Far from our initial view of d-amino acids as being limited to invertebrates, they are now considered active molecules at synapses of mammalian central and peripheral nervous systems, capable of modulating synaptic communication within neuronal networks. In particular, experimental data accumulated in the last few decades show that through the regulation of glutamatergic neurotransmission, d-serine influences the functional plasticity of cerebral circuitry throughout life. In addition, the modulation of NMDA-R-dependent signalling by d-aspartate has been demonstrated by pharmacological studies and after the targeted deletion of the d-aspartate-degrading enzyme. Considering the major contribution of the glutamatergic system to a wide range of neurological disorders such as schizophrenia, Alzheimer's disease and amyotrophic lateral sclerosis, an improved understanding of the mechanisms of d-amino-acid-dependent neuromodulation will certainly offer new insights for the development of relevant strategies to treat these neurological diseases. [ABSTRACT FROM AUTHOR]

Abstract: It was believed for long time that d-amino acids are not present in mammals. However, current technological advances and improvements in analytical instruments have enabled studies that now indicate that significant amounts of d-amino acids are present in mammals. The most abundant d-amino acids are d-serine and d-aspartate. d-Serine, which is synthesized by serine racemase and is degraded by d-amino-acid oxidase, is present in the brain and modulates neurotransmission. d-Aspartate, which is synthesized by aspartate racemase and degraded by d-aspartate oxidase, is present in the neuroendocrine and endocrine tissues and testis. It regulates the synthesis and secretion of hormones and spermatogenesis. d-Serine and d-aspartate bind to the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors and function as a coagonist and agonist, respectively. The enzymes that are involved in the synthesis and degradation of these d-amino acids are associated with neural diseases where the NMDA receptors are involved. Knockout mice for serine racemase and d-aspartate oxidase have been generated, and natural mutations in the d-amino-acid oxidase gene are present in mice and rats. These mutant animals display altered behaviors caused by enhanced or decreased NMDA receptor activity. In this article, we review currently available studies on d-amino acid metabolism in mammals and discuss analytical methods used to assay activity of amino acid racemases and d-amino-acid oxidases. [Copyright &y& Elsevier]

d-Aspartate ( d-Asp) is an endogenous substance in mammals. Degradation of d-Asp is carried out only by d-aspartate oxidase (DDO). We measured DDO activity in porcine tissues, and produced an anti-porcine DDO antibody to examine the cellular localization of DDO. All the tissues examined showed DDO activities, whereas the substrate d-Asp was not detected in kidney cortex, liver, heart, and gastric mucosa. In the kidney, intensive immunohistochemical staining for DDO was found in the epithelial cells of the proximal tubules. In the liver, the epithelial cells of interlobular bile ducts, liver sinusoid-lining cells with cytoplasmic processes, and the smooth muscle cells of arterioles were strongly stained for DDO. In the heart, cardiomyocytes and the smooth muscle cells of arterioles showed DDO-immunoreactivity. In the gastric mucosa, only the chief cells were DDO-positive. These newly identified DDO-positive cells seem to actively degrade d-Asp to prevent an excess of d-Asp from exerting harmful effects on the respective functions of porcine tissues. [ABSTRACT FROM AUTHOR]

Recent evidence has shown that -aspartate modulates hormone secretion in the vertebral neuroendocrine system. Because only -aspartate oxidase (DDO) can degrade -aspartate, we determined DDO localisation in the pituitary and pineal glands to elucidate the control mechanisms of local -aspartate concentration. Brain tissues and pituitary and pineal glands of the female pigs contained a similar DDO activity of 0.38-0.66 U/g protein. However, approximately ten-fold higher concentrations of -aspartate (0.27-0.35 μmol/g protein) were found in both glands. To determine the distribution of immunoreactive DDO, we made a rabbit polyclonal antibody specific to porcine DDO using a recombinant porcine enzyme. DDO immunoreactivity was found in the cytoplasm of a subgroup of cells in the anterior and intermediate lobes, in a part of nerve processes and terminals in the posterior lobe, and in the cytoplasm of a small group of pinealocytes. We used dual-label immunocytochemistry to determine which pituitary hormones colocalise with DDO, and whether DDO and -aspartate immunoreactivity is reciprocal. In the pituitary gland, almost all proopiomelanocortin-positive cells colocalised DDO, whereas only growth hormone-positive cells colocalised -aspartate. -Aspartate immunoreactivity was not detected where DDO immunoreactivity was found. The present study suggests that DDO plays important roles to prevent undesirable off-target action of -aspartate by strictly controlling local -aspartate concentration in the pituitary and pineal glands. [ABSTRACT FROM AUTHOR]

Abstract: d-Enantiomer amino acids, endogenously synthesized and degraded in mammals, participate in multiple developmental and physiological processes. We characterize both d-aspartate and its degradative enzyme (d-aspartate oxidase) in post-natal mouse retina. d-Aspartate attains a developmental peak of 886nmol/g dry weight at 1–2weeks post-natal while l-aspartate transiently declines, consistent with the de novo synthesis of d-aspartate. d-Aspartate is localized in many cell-types in different retinal layers, with a notable shift from outer retina to inner retina with time. d-Aspartate oxidase is localized to horizontal cells by in situ hybridization, immunohistochemistry, and histochemical staining methods. Given that final cell specification, differentiation, and synaptogenesis occur during the immediate 2-week post-natal period in the rodent retina and the dynamic appearance of d-aspartate and d-aspartate oxidase, we propose that d-aspartate plays a role in mammalian retinal development. [Copyright &y& Elsevier]

d-Aspartate oxidase (DDO, EC 1.4.3.1) catalyzes dehydrogenation of d-aspartate to iminoaspartate and the subsequent re-oxidation of reduced FAD with O2 to produce hydrogen peroxide. In the mammalian neuroendocrine system, d-aspartate, a natural substrate, plays important roles in the regulation of the synthesis and secretion of hormones. To elucidate the kinetic and structural properties of native DDO, we purified DDO from porcine kidney to homogeneity, cloned the cDNA, and overexpressed the enzyme in Escherichia coli. The purified DDO was a homotetramer with tightly-bound FAD. The enzyme consisted of 341 amino acids and had GAGVMG as the dinucleotide binding motif and a C-terminal SKL peroxisomal-targeting signal sequence. Porcine DDO showed a strong affinity for meso-tartrate (Kd = 118 μM). The oxidase exhibited pronounced substrate activation at d-aspartate and d-glutamate concentrations, [S], higher than 0.2 and 4 mM, respectively, and the [S]/v versus [S] plot showed marked downward curvature (v, the initial velocity), whereas substrate inhibition occurred with N-methyl-d-aspartate. These kinetic properties of DDO suggested that at high substrate concentrations, the FAD-reduced form of the enzyme also catalyzes the reaction: the oxidative half-reaction precedes the reductive one. The present direct approach to the analysis of non-Michaelis kinetics is indispensable for understanding the functional properties of DDO. [ABSTRACT FROM PUBLISHER]

Abstract: d-serine and d-aspartate are important regulators of mammalian physiology. d-aspartate is found in nervous and endocrine tissue, specifically in hypothalamic supraoptic and paraventricular nuclei, pituitary, and adrenal medullary cells. Endogenous d-aspartate is selectively degraded by d-aspartate oxidase. We previously reported that adult male mice lacking the gene for d-aspartate oxidase (Ddo −/− mice) display elevated concentrations of d-aspartate in several neuronal and neuroendocrine tissues as well as impaired sexual performance and altered autogrooming behaviour. In the present study, we analyzed behaviours relevant to affect, cognition, and motor control in Ddo −/− mice. Ddo −/− mice display deficits in sensorimotor gating and motor coordination as well as reduced immobility in the forced swim test. Basal corticosterone concentrations are elevated. The Ddo −/− mice have d-aspartate immunoreactive cells in the cerebellum and adrenal glands that are not observed in the wild-type mice. However, no differences in anxiety-like behaviour are detected in open field or light–dark preference tests. Also, Ddo −/− mice do not differ from wild-type mice in either passive avoidance or spontaneous alternation tasks. Although many of these behavioural deficits may be due to the lack of Ddo during development, our results are consistent with the widespread distribution of d-aspartate and the hypothesis that endogenous d-aspartate serves diverse behavioural functions. [Copyright &y& Elsevier]

• An enzymatic screening method for d- Asp-producing LAB using ChDDO was developed. • Thirteen d- Asp-producing LAB were isolated using the enzymatic screening method. • A high d- Asp-producing LAB, strain WDN19, was closely related to Lt. curvatus. • d- Asp production ability of LAB might not be related to the genera and species. 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. [ABSTRACT FROM AUTHOR]

Autism spectrum disorders (ASD) comprise a heterogeneous group of neurodevelopmental conditions characterized by impairment in social interaction, deviance in communication, and repetitive behaviors. Dysfunctional ionotropic NMDA and AMPA receptors, and metabotropic glutamate receptor 5 activity at excitatory synapses has been recently linked to multiple forms of ASD. Despite emerging evidence showing that d -aspartate and d -serine are important neuromodulators of glutamatergic transmission, no systematic investigation on the occurrence of these D-amino acids in preclinical ASD models has been carried out. Through HPLC and qPCR analyses we investigated d -aspartate and d -serine metabolism in the brain and serum of four ASD mouse models. These include BTBR mice, an idiopathic model of ASD, and Cntnap2 −/− , Shank3 −/− , and 16p11.2 +/− mice, three established genetic mouse lines recapitulating high confidence ASD-associated mutations. Biochemical and gene expression mapping in Cntnap2 −/− , Shank3 −/− , and 16p11.2 +/− failed to find gross cerebral and serum alterations in d -aspartate and d -serine metabolism. Conversely, we found a striking and stereoselective increased d -aspartate content in the prefrontal cortex, hippocampus and serum of inbred BTBR mice. Consistent with biochemical assessments, in the same brain areas we also found a robust reduction in mRNA levels of d -aspartate oxidase , encoding the enzyme responsible for d -aspartate catabolism. Our results demonstrated the presence of disrupted d -aspartate metabolism in a widely used animal model of idiopathic ASD. Overall, this work calls for a deeper investigation of D-amino acids in the etiopathology of ASD and related developmental disorders. • d -aspartate content was increased in the brain and serum of BTBR mice. • Ddo mRNA levels were reduced in prefrontal cortex and hippocampus of BTBR mice. • d -aspartate metabolism was unaltered in Cntnap2 −/− , Shank3 −/− , and 16p11.2 +/− mice. • L-glutamate levels were augmented in the serum of BTBR mice. • D-Ser metabolism was unaltered in BTBR, Cntnap2 −/− , Shank3 −/− and 16p11.2 +/− mice. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • Free D-Asp is abundant in the mammalian embryonic brain and decreases after birth. • Postnatal decline of cerebral D-Asp levels is regulated by catabolic DDO activity. • Short-term D-Asp exposure improves NMDAR-mediated brain functions. • Conversely, persistent D-Asp elevation produces NMDAR-dependent neurotoxicity. • Control of d -Asp levels by DDO has neuroprotective effects in the aging brain. [ABSTRACT FROM AUTHOR]

A mutant mouse strain ddY/DAO lacks D-amino acid oxidase activity and accumulates free neutral D-amino acids in its tissues. In this study, D-aspartate oxidase activity and D-aspartate content in the tissues of these mutant mice were compared with those of normal mice. No significant difference was observed, indicating that the metabolism of acidic D-amino acids was unaffected in the mutant. [ABSTRACT FROM AUTHOR]