D-serine Levels Research Articles

D‐Amino acids in the brain: pyridoxal phosphate‐dependent amino acid racemases and the physiology of d‐serine

Many millions of years of evolution have resulted in the virtually complete homochirality of amino acids in mammals. Not only are the amino acids that form the building blocks almost exclusively in their l-isomeric form, but free amino acids seldom appear as d-isomers. However, it has been known for a long time that d-amino acids, principally d-alanine and d-glutamic acid, are present in bacteria where they are used as building blocks for the peptidoglycan of the cell wall. Almost 60 years ago, in 1948, Sir Hans Krebs published the discovery of an enzyme that he had somewhat accidentally isolated from kidney that specifically degraded d-amino acids (but not their l counterparts). He termed this enzyme d-amino acid oxidase. However, the real function of this enzyme in vivo remained unclear. Some years later, another flavoenzyme, d-aspartate oxidase, was also discovered in mammals, and its specificity was shown to be focused on dicarboxylic d-amino acids. The identification of free d-amino acids in several tissues and in the blood of mammals finally provided a rationale for the widespread occurrence of these oxidases. Initially, the mammalian d-amino acids were thought to arise from endogenous microbial flora, from ingestion in the diet, or from spontaneous racemization of l-amino acids incorporated into polypeptides during aging. With the improvement in HPLC techniques, the unambiguous detection of d-amino acids has been possible. The three most abundant d-amino acids in mammalian brain are d-alanine, d-serine and d-aspartic acid. For instance, d-alanine has been found in rat brain as well as in the pituitary gland. d-Serine is also present in the nervous system in high concentrations, mostly located in the grey matter, in the hippocampus, in the anterior olfactory nucleus and in the amygdala. High levels of d-aspartic acid also occur in the brain and endocrine glands, such as pineal, adrenal and pituitary. The most important breakthrough in the field is that of Wolosker, Snyder and coworkers who, using dozens of rat brains, were able to isolate an enzymatic activity that could convert l-serine into d-serine. Elegant experiments allowed them to clone the structural gene and demonstrate that the enzyme was a pyridoxal phosphate (PLP)-containing racemase. This mammalian serine racemase could increase the intersynaptic levels of d-serine, a molecule that used the so-called glycine site within the N-methyl d-aspartate (NMDA) receptor. Less is known about the enzyme(s) responsible for the synthesis of d-aspartic acid or d-alanine. The observation that amino-oxyacetic acid, a potent inhibitor of PLP-dependent enzymes, could abrogate the release of d-aspartic acid in primary neuronal cultures indicates that aspartate racemase might also be a PLP-containing enzyme. So far the identification of the racemases responsible for the synthesis of d-aspartic acid and d-alanine has been elusive. In these three reviews we initially analyze the physiology of d-serine in the brain, notably the localization and regulation of brain serine racemase, both in neurotransmission and in neurodegeneration. In the second article we describe how PLP-dependent enzymes perform the racemization reaction, both in microorganisms and in higher organisms. Finally, in a third review, the regulation of serine racemase by divalent cations, nucleotides and interacting proteins is also addressed.

WHAT ARE THE RULES? COULD WE HAVE AVOIDED UNPLEASANT SURPRISES?

N-Methyl-d-aspartate (NMDA) receptors, which play an important role in neuronal excitotoxicity, require not only agonists at the glutamate-binding site but also co-agonists at the glycine site for their activation. Here we examined the role of endogenous agonists at the glycine site of NMDA receptors in excitotoxic retinal damage in vivo. To quantify the number of surviving retinal ganglion cells (RGCs), we injected a retrograde tracer, fluoro-gold, into the superior colliculus bilaterally and subsequently counted RGCs on whole-mounted retinas. Co-injection of 5,7-dichlorokynurenic acid (300 nmol), a competitive antagonist at the glycine site of NMDA receptors, rescued RGCs from damage induced by 200 nmol NMDA. On the other hand, RGC death induced by 20 nmol NMDA was enhanced by addition of glycine (10 nmol), d-serine (10 nmol) or a competitive glycine transporter-1 inhibitor, sarcosine (0.3 or 3 nmol). Moreover, application of d-serine-degrading enzyme, d-amino acid oxidase (30 mU), partially suppressed RGC death induced by 20 nmol NMDA. These results suggest that the severity of excitotoxic retinal damage in vivo depends on the levels of both glycine and d-serine.

Synthesis and Biological Evaluation of d-Amino Acid Oxidase Inhibitors

D-amino acid oxidase (DAAO) catalyzes the oxidation of D-amino acids including d-serine, a full agonist at the glycine site of the NMDA receptor. A series of benzo[ d]isoxazol-3-ol derivatives were synthesized and evaluated as DAAO inhibitors. Among them, 5-chloro-benzo[ d]isoxazol-3-ol (CBIO) potently inhibited DAAO with an IC50 in the submicromolar range. Oral administration of CBIO in conjunction with d-serine enhanced the plasma and brain levels of d-serine in rats compared to the oral administration of d-serine alone.

Astroglia: Not Just Glue

Astroglia: Not Just Glue

The discovery of fused pyrrole carboxylic acids as novel, potent d-amino acid oxidase (DAO) inhibitors

The ‘NMDA hypofunction hypothesis of schizophrenia’ can be tested in a number of ways. DAO is the enzyme primarily responsible for the metabolism of d-serine, a co-agonist for the NMDA receptor. We identified novel DAO inhibitors, in particular, acid 1, which demonstrated moderate potency for DAO in vitro and ex vivo, and raised plasma d-serine levels after dosing ip to rats. In parallel, analogues were prepared to survey the SARs of 1.

Mice lacking d-amino acid oxidase activity exhibit marked reduction of methamphetamine-induced stereotypy

The behavioral effects induced by methamphetamine (5.0 mg/kg) were compared in the mutant mice lacking d-amino acid oxidase activity and normal mice. The mutant mice exhibited marked decline in the methamphetamine-induced stereotypy compared to the normal mice, whereas the mutant mice displayed a drastic augmentation in the locomotor activity evoked by methamphetamine compared to the normal mice. Because the d-serine levels in the brain of the mutant mice are significantly higher than those in the normal mice, the enhanced d-serine in the brain of the mutant mice could antagonize the methamphetamine-induced stereotypy via the N-methyl- d-aspartate receptors.

D-serine serum level - a marker of glutamatergic dysfunction in schizophrenia

Background:D-serine acts as an endogenous co-agonist at the glycine modulatory site of the NMDA receptor. Significantly decreased D-serine serum levels were reported in patients with schizophrenia in comparison to healthy control subjects. D-serine improved positive and negative symptoms in patients with schizophrenia treated with antipsychotics. We hypothesized that D-serine serum level might be associated with specific characteristics of psychopathology in schizophrenia.Methods:We enrolled fifty patients with schizophrenia into the study. Positive and Negative Syndrom Scale (PANSS) and The Scale for the Assessment of Negative Symptoms (SANS) were used to assess the symptoms of schizophrenia. D-serine serum levels were measured by High Performance Liquid Chromatography.Results:D-serine serum levels were not associated with PANSS and SANS total and subscales scores in the population of fifty patients. We demonstrated only mild insignificant linear association of PANSS score with D-serine serum level (r=0.20) in the group of men (n=33). The mild insignificant inverse correlation was found in the group of women (n=17) between the total PANSS (r=-0.35) or SANS score (r=-0.30) and D-serine serum level.Conclusion:We assumed that various biochemical and clinical profiles could lead to identification of specific subtypes of schizophrenia. However, we did not find any significant association between serum D-serine and clinical symptoms in this study. D-serine serum levels had a strong trend to be lower among female patients with schizophrenia as compared to men. The role of gender in the glutamatergic dysfunction associated with schizophrenia deserves further attention.

Characterization of a Yeastd-Amino Acid Oxidase Microbiosensor ford-Serine Detection in the Central Nervous System

d-Serine is an endogenous ligand for N-methyl-d-aspartate (NMDA) receptors, and alterations in its concentration have been related to several brain disorders, especially schizophrenia. It is therefore an important target neuromodulator for the pharmaceutical industry. To monitor d-serine levels in vivo, we have developed a microbiosensor based on cylindrical platinum microelectrodes, covered with a membrane of poly-m-phenylenediamine (PPD) and a layer of immobilized d-amino acid oxidase from the yeast Rhodotorula gracilis (RgDAAO). By detecting the hydrogen peroxide produced by enzymatic degradation of d-serine, this microbiosensor shows a detection limit of 16 nM and a mean response time of 2 s. Interferences by ascorbic acid, uric acid, l-cysteine, and by biogenic amines and their metabolites are rejected at more than 97% by the PPD layer. Although several d-amino acids are potential substrates for RgDAAO, d-serine was the only endogenous substrate present in sufficient concentration to be detected by our microbiosensor in the central nervous system. When implanted in the cortex of anesthetized rats, this microbiosensor detected the increase in concentration of d-serine resulting from its diffusion across the blood-brain barrier after an intraperitoneal injection. This new device will make it possible to investigate in vivo the variations in d-serine concentrations occurring under normal and pathological conditions and to assess the pharmacological potency of new drugs designed to impact d-serine metabolism.

Modulation of d-Serine Levels in Brains of Mice Lacking PICK1

D-serine is an endogenous coagonist of the N-methyl-D-aspartate subtype glutamate receptor. Genetic association studies have implicated genes coding for enzymes associated with D-serine metabolism in schizophrenia and bipolar disorder. Protein expression of serine racemase (SR) and its binding partner, protein interacting with C-kinase (PICK1), were examined by Western blotting in brains from wildtype and PICK1 knockout mice. Levels of D-serine in wildtype and PICK1 mice were also examined by an established high-pressure liquid chromatography protocol. Expression of SR and PICK1 proteins was developmentally regulated. Although no change was observed in the level of SR protein, levels of D-serine were selectively decreased in the forebrain of neonatal PICK1 knockout mice, compared with those in wildtype mice. PICK1 may be involved in the regulation of brain D-serine levels and SR in a spatially and temporally specific manner.

Long-Term Treatment With Morphine Increases the D-Serine Content in the Rat Brain by Regulating the mRNA and Protein Expressions of Serine Racemase and D-Amino Acid Oxidase

Recent studies indicate that an endogenous co-agonist for an N-methyl-D-aspartate (NMDA) receptor–related glycine site, D-serine, is synthesized by serine racemase and is metabolized by D-amino acid oxidase (DAO) and that acute treatment with morphine augments the gene expression of serine racemase and DAO in rat brain. To further elucidate the mechanism underlying the activation of NMDA receptors following chronic opioid administration, we have evaluated the effects of the chronic administration of morphine on the mRNA and protein expressions of serine racemase and DAO and on the contents of D-serine in several areas of the rat brain. Repeated administration of morphine for 30 days produced a significant augmentation of both the mRNA and protein expressions of serine racemase in all the brain regions, whereas no significant change in the protein expression of DAO was observed in all the brain regions. Furthermore, the chronic administration caused a slight but significant elevation in the concentration of D-serine in the cortex, striatum, and hippocampus. These results indicate the elevated D-serine level following the chronic morphine treatment could at least in part be involved in the activation of NMDA receptors via the glycine site.

Serine racemase expression and d‐serine content are developmentally regulated in neuronal ganglion cells of the retina

D-Serine, the endogenous ligand for the glycine modulatory binding site of the NMDA receptor, and serine racemase, the enzyme that converts L-serine to D-serine, have been reported in vertebrate retina; initial reports suggested that localization was restricted to Müller glial cells. Recent reports, in which D-serine and serine racemase were detected in neurons of the brain, prompted the present investigation of neuronal expression of D-serine and serine racemase in retina and whether expression patterns were developmentally regulated. RT-PCR, in situ hybridization, western blotting, immunohistochemistry, and immunocytochemical methods were used to localize D-serine and serine racemase in intact retina obtained from 1 to 3 day, 3 week, and 18 week mouse retinas and in primary ganglion cells harvested by immunopanning from neonatal mouse retina. Results of these analyses revealed robust expression of D-serine and serine racemase in ganglion cells, both in intact retina and in cultured cells. The levels appear to be developmentally regulated with D-serine levels being quite high in ganglion cells of neonatal retinas and decreasing rapidly postnatally. Serine racemase levels are also developmentally regulated, with high levels detected during the early postnatal period, but diminishing considerably in the mature retina. This represents the first report of neuronal expression of D-serine and serine racemase in the vertebrate retina and suggests an important contribution of neuronal D-serine during retinal development.

In vitro and in vivo pharmacological profile of AS057278, a selective d-amino acid oxidase inhibitor with potential anti-psychotic properties

Non-competitive N-methyl-d-aspartate (NMDA) blockers induce schizophrenic-like behavior in healthy volunteers and exacerbate symptomatology in schizophrenic patients. Hence, a compound able to enhance NMDA neurotransmission by increasing levels of d-serine, an endogenous full agonist at the glycine site of the NMDA receptors, could have anti-psychotic activity. One way to increase d-serine levels is the inhibition of d-amino acid oxidase (DAAO), the enzyme responsible for d-serine oxidation. Indeed AS057278, a potent in vitro (IC50=0.91 μM) and ex vivo (ED50=2.2–3.95 μM) DAAO inhibitor, was able to increase d-serine fraction in rat cortex and midbrain (10 mg/kg i.v.). AS057278 was able to normalize phencyclidine (PCP)-induced prepulse inhibition after acute (80 mg/kg) and chronic (20 mg/kg b.i.d.) oral administration in mice. Finally, AS057278 after oral chronic treatment (10 mg/kg b.i.d.) was able to normalize PCP-induced hyperlocomotion. These results suggest that AS057278 has the potential to anti-psychotic action toward both cognitive and positive symptoms of schizophrenia.

Contribution of endogenous glycine and d-serine to excitotoxic and ischemic cell death in rat cerebrocortical slice cultures

N-methyl- d-aspartate (NMDA) receptors, whose activation requires glycine site stimulation, play crucial roles in various physiological and pathological conditions in the brain. We investigated the regulatory roles of potential endogenous glycine site agonists, glycine and d-serine, in excitotoxic and ischemic cell death in the cerebral cortex. Cytotoxicity of NMDA on rat cerebrocortical slice cultures was potentiated by addition of glycine or d-serine. In contrast, cell death induced by oxygen/glucose deprivation (OGD) was not affected by exogenous glycine or d-serine, although blockade of NMDA receptors by MK-801 abolished cell death. In addition, higher concentrations of 2,7-dichlorokynurenic acid (DCKA), a competitive glycine site antagonist, were required to suppress OGD-induced cell death than those to suppress NMDA cytotoxicity. We also found that OGD triggered a robust increase in extracellular glycine. A glycine transporter blocker ALX 5407 increased the extracellular level of glycine, and the protective effect of DCKA against NMDA cytotoxicity was diminished in the presence of ALX 5407. Sensitivity of NMDA cytotoxicity to DCKA was also diminished by l-serine that increased the extracellular level of d-serine. These results indicate that both glycine and d-serine can act as endogenous ligands for NMDA receptor glycine site in the cerebral cortex, and that endogenous glycine may saturate the glycine site under ischemic conditions. The present findings are important for the interpretation of the mechanisms of NMDA and OGD cytotoxicity.

Induction of Serine Racemase Expression and D-Serine Release from Microglia by Secreted Amyloid Precursor Protein (sAPP)

Alzheimer's disease (AD) involves neuronal loss and reduction of synaptic density in specific brain region. Some of the neuronal deaths are associated with excitotoxicity. We previously reported that amyloid beta-peptide (Abeta) induced release of N-methyl-D-aspartate receptor (NMDA-R) co-agonists, including glutamate and D-serine. The induction of D-serine production by Abeta involves transcriptional and/or translational regulation of serine racemase gene. Similarly, we report here that conditioned medium from microglia treated with secreted amyloid precursor protein (sAPP) contained elevated levels of D-serine. In microglia, sAPP increased the steady-state dimeric protein level of serine racemase. Promoter-reporter and mRNA analyses suggested that serine racemase is transcriptionally induced by sAPP. These data extend the link between excitotoxicity and neuroinflammation. D-serine may cooperate with glutamate to link neuroinflammation with excitotoxicity, suggesting a pathogenic mechanism applicable to neuronal death in AD and other neurodegenerative diseases.

Physiological functions of D-amino acid oxidases: from yeast to humans.

D-Amino acid oxidase (DAAO) is a FAD-containing flavoenzyme that catalyzes the oxidative deamination of D-isomers of neutral and polar amino acids. This enzymatic activity has been identified in most eukaryotic organisms, the only exception being plants. In the various organisms in which it does occur, DAAO fulfills distinct physiological functions: from a catabolic role in yeast cells, which allows them to grow on D-amino acids as carbon and energy sources, to a regulatory role in the human brain, where it controls the levels of the neuromodulator D-serine. Since 1935, DAAO has been the object of an astonishing number of investigations and has become a model for the dehydrogenase-oxidase class of flavoproteins. Structural and functional studies have suggested that specific physiological functions are implemented through the use of different structural elements that control access to the active site and substrate/product exchange. Current research is attempting to delineate the regulation of DAAO functions in the contest of complex biochemical and physiological networks.

NMDA receptor in conditioned flavor-taste preference learning: Blockade by MK-801 and enhancement by D-cycloserine

Conditioned flavor-taste preference (CFTP) is a robust form of learning in which animals acquire a preference for a flavor (e.g. Kool-Aid) previously mixed with a highly preferred tastant (e.g. fructose) over a flavor previously mixed with a less-preferred tastant (e.g. saccharin). Here, the role of the N-methyl- D-aspartate (NMDA) glutamate–glycine receptor (NR) was probed using systemic MK-801, a non-competitive antagonist, and D-cycloserine (DCS), a glycine agonist. Rats were injected with MK-801 (100 μg/kg) or vehicle 30 min prior to a daily 2-h conditioning session with 1-bottle access to a Kool-Aid flavor (grape or cherry) mixed with either 8% fructose (CS+/F) or 0.2% saccharin (CS−/S). CFTP expression was measured in 2-bottle preference tests between the Kool-Aid flavors mixed with 0.2% saccharin (CS+/S vs. CS−/S). While vehicle-treated rats acquired a preference for CS+/S over CS−/S, MK-801 prior to conditioning completely blocked CFTP learning. The effect of MK-801 was specific to CFTP acquisition, because follow-up experiments demonstrated that MK-801 did not induce a conditioned taste aversion, cause state-dependent learning, or affect CFTP expression. In a second approach, rats were injected with DCS (15 mg/kg) 60 min prior to daily conditioning. In contrast to MK-801, administration of DCS prior to conditioning enhanced CFTP learning (but not reversal conditioning). These results demonstrate that NR neurotransmission is critical for CFTP learning. Furthermore, enhancement of CFTP learning by DCS suggests that endogenous levels of glycine or D-serine may be a limiting factor in CFTP learning.

A CSF and postmortem brain study of d-serine metabolic parameters in schizophrenia

Clinical trials demonstrated that d-serine administration improves schizophrenia symptoms, raising the possibility that altered levels of endogenous d-serine may contribute to the N-methyl d-aspartate receptor hypofunction thought to play a role in the disease. We hypothesized that cerebro-spinal fluid (CSF) d-serine levels are decreased in the patients due to reduced synthesis and/or increased degradation in brain. We now monitored amino acid levels in CSF from 12 schizophrenia patients vs. 12 controls and in postmortem parietal-cortex from 15 control subjects and 15 each of schizophrenia, major-depression and bipolar patients. In addition, we monitored postmortem brain serine racemase and d-amino acid oxidase protein levels by Western-blot analysis. We found a 25% decrease in d-serine levels and d/ l-serine ratio in CSF of schizophrenia patients, while parietal-cortex d-serine was unaltered. Levels of l-serine, l-glutamine and l-glutamate were unaffected. Frontal-cortex (39%) and hippocampal (21%) serine racemase protein levels and hippocampal serine racemase/ d-amino acid oxidase ratio (34%) were reduced. Hippocampal d-amino-acid-oxidase protein levels significantly correlated with duration of illness ( r = 0.6, p = 0.019) but not age. d-amino acid oxidase levels in patients with DOI > 20 years were 77% significantly higher than in the other patients and controls. Our results suggest that reduced brain serine racemase and elevated d-amino acid oxidase protein levels may contribute to the lower CSF d-serine levels in schizophrenia.

A Genetic Variant of the Serine Racemase Gene Is Associated with Schizophrenia

Serine racemase (SRR) is a brain-enriched enzyme that converts L-serine to D-serine, which acts as an endogenous ligand of N-methyl D-aspartate (NMDA) receptors. Dysfunction of SRR may reduce the function of NMDA receptors and susceptibility to schizophrenia. We genotyped three single-nucleotide polymorphisms (SNPs) of the 5' region of the SRR gene in 525 patients with schizophrenia and 524 healthy controls. Effects of SNPs on the promoter activity and on serum levels of total and D-serine were examined. We found a significant excess of the IVS1a+465C allele of the SRR gene in schizophrenia, especially in the paranoid subtype (p = .0028). A reporter assay showed that the IVS1a+465C allele had 60% lower promoter activity than did the IVS1a+465G allele. The IVS1a+465C allele of the SRR gene, which reduces expression of the gene, is a risk factor for schizophrenia, especially the paranoid subtype.

Effects of MK-801 on the expression of serine racemase and d-amino acid oxidase mRNAs and on the d-serine levels in rat brain

We have investigated the acute effects of the increasing doses of non-competitive N-methyl- d-aspartate receptor antagonist MK-801 (0.2–1.6 mg/kg) on the expression of serine racemase and d-amino acid oxidase (DAO) mRNAs in several brain areas of rats. We have also evaluated the effects of the chronic administration of MK-801 (0.4 mg/kg) on the gene expression of serine racemase and DAO and on the d-serine concentrations. A dose-dependent augmentation of the expression of serine racemase mRNA was seen in most brain areas at both 1 and 4 h after the administration. In contrast, a drastic decline in the expression of DAO mRNA was observed in most brain areas 1 h after the MK-801 administration, whereas a dose-dependent elevation in the expression of DAO mRNA was observed in most brain areas 4 h after the administration. The chronic MK-801 administration produced a significant increase in the expression of serine racemase mRNA in almost all brain areas, whereas no significant changes were found in the level of DAO mRNA in most brain areas. In addition, the chronic administration caused a slight but significant elevation in the concentrations of d-serine in the cortex and striatum. These present findings indicate that increasing the serine racemase mRNA and no changes in the DAO mRNA after the chronic administration could contribute to the elevation of the d-serine level in the forebrain, and that serine racemase and DAO could play an important role in the regulation of N-methyl- d-aspartate receptors via the d-serine metabolism.

Contribution of endogenous glycine site NMDA agonists to excitotoxic retinal damage in vivo

N-Methyl- d-aspartate (NMDA) receptors, which play an important role in neuronal excitotoxicity, require not only agonists at the glutamate-binding site but also co-agonists at the glycine site for their activation. Here we examined the role of endogenous agonists at the glycine site of NMDA receptors in excitotoxic retinal damage in vivo. To quantify the number of surviving retinal ganglion cells (RGCs), we injected a retrograde tracer, fluoro-gold, into the superior colliculus bilaterally and subsequently counted RGCs on whole-mounted retinas. Co-injection of 5,7-dichlorokynurenic acid (300 nmol), a competitive antagonist at the glycine site of NMDA receptors, rescued RGCs from damage induced by 200 nmol NMDA. On the other hand, RGC death induced by 20 nmol NMDA was enhanced by addition of glycine (10 nmol), d-serine (10 nmol) or a competitive glycine transporter-1 inhibitor, sarcosine (0.3 or 3 nmol). Moreover, application of d-serine-degrading enzyme, d-amino acid oxidase (30 mU), partially suppressed RGC death induced by 20 nmol NMDA. These results suggest that the severity of excitotoxic retinal damage in vivo depends on the levels of both glycine and d-serine.