Synaptic and extrasynaptic NMDA receptors are gated by different endogenous coagonists.

N-methyl-d-aspartate receptors (NMDARs) are located in neuronal cell membranes at synaptic and extrasynaptic locations, where they are believed to mediate distinct physiological and pathological processes. Activation of NMDARs requires glutamate and a coagonist whose nature and impact on NMDAR physiology remain elusive. We report that synaptic and extrasynaptic NMDARs are gated by different endogenous coagonists, d-serine and glycine, respectively. The regionalized availability of the coagonists matches the preferential affinity of synaptic NMDARs for d-serine and extrasynaptic NMDARs for glycine. Furthermore, glycine and d-serine inhibit NMDAR surface trafficking in a subunit-dependent manner, which is likely to influence NMDARs subcellular location. Taking advantage of this coagonist segregation, we demonstrate that long-term potentiation and NMDA-induced neurotoxicity rely on synaptic NMDARs only. Conversely, long-term depression requires both synaptic and extrasynaptic receptors. Our observations provide key insights into the operating mode of NMDARs, emphasizing functional distinctions between synaptic and extrasynaptic NMDARs in brain physiology.

Dopaminergic neuromodulation of prefrontal cortex activity requires the NMDA receptor coagonist d-serine.

Prefrontal control of cognitive functions critically depends upon glutamatergic transmission and N-methyl D-aspartate (NMDA) receptors, the activity of which is regulated by dopamine. Yet whether the NMDA receptor coagonist d-serine is implicated in the dopamine-glutamate dialogue in the prefrontal cortex (PFC) and other brain areas remains unexplored. Here, using electrophysiological recordings, we show that d-serine is required for the fine-tuning of glutamatergic neurotransmission, neuronal excitability, and synaptic plasticity in the PFC through the actions of dopamine at D1 and D3 receptors. Using in vivo microdialysis, we show that D1 and D3 receptors exert a respective facilitatory and inhibitory influence on extracellular levels and activity of d-serine in the PFC, with actions expressed primarily via the cAMP/protein kinase A (PKA) signaling cascade. Further, using functional magnetic resonance imaging (fMRI) and behavioral assessment, we show that d-serine is required for the potentiation of cognition by D3R blockade as revealed in a test of novel object recognition memory. Collectively, these results unveil a key role for d-serine in the dopaminergic neuromodulation of glutamatergic transmission and PFC activity, findings with clear relevance to the pathogenesis and treatment of diverse brain disorders involving alterations in dopamine-glutamate cross-talk.

Antimicrobial D-amino acid oxidase-derived peptides specify gut microbiota.

The flavoenzyme D-amino acid oxidase (DAAO) is deputed to the degradation of D-enantiomers of amino acids. DAAO plays various relevant physiological roles in different organisms and tissues. Thus, it has been recently suggested that the goblet cells of the mucosal epithelia secrete into the lumen of intestine, a processed and active form of DAAO that uses the intestinal D-amino acids to generate hydrogen peroxide (H2O2), an immune messenger that helps fighting gut pathogens, and by doing so controls the homeostasis of gut microbiota. Here, we show that the DAAO form lacking the 1-16 amino acid residues (the putative secretion signal) is unstable and inactive, and that DAAO is present in the epithelial layer and the mucosa of mouse gut, where it is largely proteolyzed. In silico predicted DAAO-derived antimicrobial peptides show activity against various Gram-positive and Gram-negative bacteria but not on Lactobacilli species, which represent the commensal microbiota. Peptidomic analysis reveals the presence of such peptides in the mucosal fraction. Collectively, we identify a novel mechanism for gut microbiota selection implying DAAO-derived antimicrobial peptides which are generated by intestinal proteases and that are secreted in the gut lumen. In conclusion, we herein report an additional, ancillary role for mammalian DAAO, unrelated to its enzymatic activity.

L-serine synthesis via the phosphorylated pathway in humans.

L-serine is a nonessential amino acid in eukaryotic cells, used for protein synthesis and in producing phosphoglycerides, glycerides, sphingolipids, phosphatidylserine, and methylenetetrahydrofolate. Moreover, L-serine is the precursor of two relevant coagonists of NMDA receptors: glycine (through the enzyme serine hydroxymethyltransferase), which preferentially acts on extrasynaptic receptors and D-serine (through the enzyme serine racemase), dominant at synaptic receptors. The cytosolic "phosphorylated pathway" regulates de novo biosynthesis of L-serine, employing 3-phosphoglycerate generated by glycolysis and the enzymes 3-phosphoglycerate dehydrogenase, phosphoserine aminotransferase, and phosphoserine phosphatase (the latter representing the irreversible step). In the human brain, L-serine is primarily found in glial cells and is supplied to neurons for D-serine synthesis. Serine-deficient patients show severe neurological symptoms, including congenital microcephaly, psychomotor retardation, and intractable seizures, thus highlighting the relevance of de novo production of this amino acid in brain development and morphogenesis. Indeed, the phosphorylated pathway is strictly linked to cancer. Moreover, L-serine has been suggested as a ready-to-use treatment, as also recently proposed for Alzheimer's disease. Here, we present our current state of knowledge concerning the three mammalian enzymes of the phosphorylated pathway and known mutations related to pathological conditions: although the structure of these enzymes has been solved, how enzyme activity is regulated remains largely unknown. We believe that an in-depth investigation of these enzymes is crucial to identify the molecular mechanisms involved in modulating concentrations of the serine enantiomers and for studying the interplay between glial and neuronal cells and also to determine the most suitable therapeutic approach for various diseases.

An antibody-based enzymatic therapy for cancer treatment: The selective localization of D-amino acid oxidase to EDA fibronectin.

In order to generate an antibody directed enzyme prodrug therapy, here we designed a chimeric protein by fusing the F8 antibody that recognizes the EDA of fibronectin (expressed on the tumor neovasculature) and an evolved variant of the ROS-generating enzyme D-amino acid oxidase (DAAO). The F8(scFv)-DAAO-Q144R recombinant protein is expressed by both CHO-S and E. coli cells. The F8(scFv)-DAAO-Q144R from E. coli cells is fully soluble, shows a high specific activity, is more thermostable in blood than the native DAAO, possesses a binding affinity for EDA well suited for efficient tumor accumulation, and localizes in tumor tissues. Notably, the F8(scFv)-DAAO-Q144R conjugate generates a stronger cytotoxicity to tumor cells than the native enzyme, especially when an inhibitor of heme oxygenase-1 (HO-1) is used, making it a promising candidate for a selective antitumor oxidative therapy controlled by the substrate addition, in the so called "activity on demand", thus sparing normal tissue from damage.

Direct chromatographic methods for enantioresolution of amino acids: recent developments.

α-Amino acids are present in two opposite configurations due to the presence of a central carbon atom which is a chiral center. While L-amino acids are present in large amount in nature, only tiny quantities of their D-enantiomers exist. For a long time, D-amino acids have been considered of bacterial origin only, but recently we realized that they are present in all living organisms: notably, D-amino acids play specific and relevant functions in the different organisms. Detection and quantification of D-amino acids are mandatory to shed light on their physiological roles, especially related to foods and human health. Chromatographic techniques are among the most commonly used analytical methods for the enantioseparation of amino acids. Here, we revised the latest improvements in chromatographic direct methodologies based on chiral selectors and aimed to improve analytical speed, sensitivity, robustness, and reproducibility. While current methods are well suited for D-amino acid analysis in foodstuffs and pharmaceuticals, further improvements seem required for their simultaneous, fast and sensitive detection in biological fluids, an emerging field since D-amino acids have been proposed as biomarkers of different and relevant human pathologic states.

Human d-amino acid oxidase: The inactive G183R variant.

In the brain, the enzyme d-amino acid oxidase (DAAO) catalyzes the oxidative deamination of d-serine, a main positive modulator of the N-methyl-d-aspartate subtype of glutamate receptors (NMDAR). Dysregulation in d-serine signaling is implicated in the NMDAR dysfunctions observed in various brain diseases, such as amyotrophic lateral sclerosis, Alzheimer's disease, schizophrenia. A strain of ddY mice lacking DAAO activity due to the G181R substitution (DAAOG181R mice) and exhibiting increased d-serine concentration as compared to wild-type mice shows altered pain response, improved adaptative learning and cognitive functions, and larger hippocampal long-term potentiation. In past years, this mice line has been used to shed light on physiological and pathological brain functions related to NMDAR. Here, we decided to introduce the corresponding substitution in human DAAO (hDAAO). The recombinant G183R hDAAO is produced as an inactive apoprotein: the substitution alters the protein conformation that negatively affects the ability to bind the flavin cofactor in the orientation required for hydride-transfer during catalysis. At the cellular level, the overexpressed G183R hDAAO is not fully targeted to peroxisomes, forms protein aggregates showing a strong colocalization with ubiquitin, and significantly (7-fold) increases both the d-serine cellular concentration and the D/(D+L)-serine ratio. Taken together, our investigation warrants caution in using DAAOG181R mice: the abolition of enzymatic activity is coupled to DAAO aggregation, a central process in different pathological conditions. The effect due to G181R substitution in DAAO could be misleading: the effects due to impairment of d-serine degradation overlap with those related to aggregates accumulation.

Identity of the NMDA receptor coagonist is synapse specific and developmentally regulated in the hippocampus.

NMDA receptors (NMDARs) require the coagonists D-serine or glycine for their activation, but whether the identity of the coagonist could be synapse specific and developmentally regulated remains elusive. We therefore investigated the contribution of D-serine and glycine by recording NMDAR-mediated responses at hippocampal Schaffer collaterals (SC)-CA1 and medial perforant path-dentate gyrus (mPP-DG) synapses in juvenile and adult rats. Selective depletion of endogenous coagonists with enzymatic scavengers as well as pharmacological inhibition of endogenous D-amino acid oxidase activity revealed that D-serine is the preferred coagonist at SC-CA1 mature synapses, whereas, unexpectedly, glycine is mainly involved at mPP-DG synapses. Nevertheless, both coagonist functions are driven by the levels of synaptic activity as inferred by recording long-term potentiation generated at both connections. This regional compartmentalization in the coagonist identity is associated to different GluN1/GluN2A to GluN1/GluN2B subunit composition of synaptic NMDARs. During postnatal development, the replacement of GluN2B- by GluN2A-containing NMDARs at SC-CA1 synapses parallels a change in the identity of the coagonist from glycine to D-serine. In contrast, NMDARs subunit composition at mPP-DG synapses is not altered and glycine remains the main coagonist throughout postnatal development. Altogether, our observations disclose an unprecedented relationship in the identity of the coagonist not only with the GluN2 subunit composition at synaptic NMDARs but also with astrocyte activity in the developing and mature hippocampus that reconciles the complementary functions of D-serine And Glycine In Modulating Nmdars During The Maturation Of Tripartite Glutamatergic Synapses.

Age-Related Changes in D-Aspartate Oxidase Promoter Methylation Control Extracellular D-Aspartate Levels and Prevent Precocious Cell Death during Brain Aging.

The endogenous NMDA receptor (NMDAR) agonist D-aspartate occurs transiently in the mammalian brain because it is abundant during embryonic and perinatal phases before drastically decreasing during adulthood. It is well established that postnatal reduction of cerebral D-aspartate levels is due to the concomitant onset of D-aspartate oxidase (DDO) activity, a flavoenzyme that selectively degrades bicarboxylic D-amino acids. In the present work, we show that d-aspartate content in the mouse brain drastically decreases after birth, whereas Ddo mRNA levels concomitantly increase. Interestingly, postnatal Ddo gene expression is paralleled by progressive demethylation within its putative promoter region. Consistent with an epigenetic control on Ddo expression, treatment with the DNA-demethylating agent, azacitidine, causes increased mRNA levels in embryonic cortical neurons. To indirectly evaluate the effect of a putative persistent Ddo gene hypermethylation in the brain, we used Ddo knock-out mice (Ddo(-/-)), which show constitutively suppressed Ddo expression. In these mice, we found for the first time substantially increased extracellular content of d-aspartate in the brain. In line with detrimental effects produced by NMDAR overstimulation, persistent elevation of D-aspartate levels in Ddo(-/-) brains is associated with appearance of dystrophic microglia, precocious caspase-3 activation, and cell death in cortical pyramidal neurons and dopaminergic neurons of the substantia nigra pars compacta. This evidence, along with the early accumulation of lipufuscin granules in Ddo(-/-) brains, highlights an unexpected importance of Ddo demethylation in preventing neurodegenerative processes produced by nonphysiological extracellular levels of free D-aspartate.

G72 primate-specific gene: a still enigmatic element in psychiatric disorders.

Numerous studies have demonstrated a link between genetic markers on chromosome 13 and schizophrenia, bipolar affective disorder, and other psychiatric phenotypes. The G72/G30 genes (transcribed in opposite directions) are located on chromosome 13q33, a region demonstrating strong evidence for linkage with various neuropsychiatric disorders. G72/G30 was identified in 2002 as a schizophrenia susceptibility locus; however, subsequent association studies did not reach consensus on single SNPs within the locus. Simultaneously, a new vision for the genetic architecture of psychiatric disorders suggested that schizophrenia was a quantitative trait, therefore ascribable to potentially hundreds of genes and subjected to the vagaries of the environment. The main protein product of G72 gene is named pLG72 or D-amino acid oxidase activator DAOA (153 amino acids) and its function is still debated. Functional analyses, also showing controversial results, indicate that pLG72 contributes to N-methyl-D-aspartate receptor modulation by affecting activity of the flavoprotein D-amino acid oxidase, the enzyme responsible for degrading the neuromodulator D-serine. In this review we, for the first time, summarize findings from molecular genetic linkage and association studies concerning G72 gene, cellular and molecular studies on pLG72, and investigations performed on G72/G30 transgenic mice. This will help elucidate the role of psychosis susceptibility genes, which will have a major impact on our understanding of disease pathophysiology and thus change classification and treatment.

Novel insights into renal D-amino acid oxidase accumulation: propiverine changes DAAO localization and peroxisomal size in vivo.

Chronic exposure to propiverine, a frequently prescribed pharmaceutical for treatment of overactive bladder and incontinence, provokes massive protein accumulation in the cytosol and nucleus of renal proximal tubule epithelial cells in rats. Previously, the accumulating protein was identified as D-amino acid oxidase (DAAO), a peroxisomal flavoenzyme expressed in kidney, liver and brain. The cellular mechanism of propiverine-induced DAAO accumulation, however, remains unexplained and poorly characterized. Therefore, to further increase the understanding of DAAO accumulation in rat kidney, this study aimed to characterize DAAO accumulations using differential immunofluorescent staining of rat kidney sections as well as in vitro binding analyses and proteasomal activity studies. We demonstrated that propiverine is neither a ligand of DAAO nor an inhibitor of the proteasome in vitro. However, propiverine treatment resulted in a significant decrease of peroxisomal size in rat proximal tubule epithelial cells. Moreover, peroxisomal catalase also accumulated in the cytosol and nuclei of propiverine-treated rats concurrently with DAAO. Taken together, our study indicates that propiverine treatment affects the trafficking and/or degradation of peroxisomal proteins such as DAAO and catalase by a so far unique and unknown mechanism.

Understanding renal nuclear protein accumulation: an in vitro approach to explain an in vivo phenomenon.

Proper subcellular trafficking is essential to prevent protein mislocalization and aggregation. Transport of the peroxisomal enzyme D-amino acid oxidase (DAAO) appears dysregulated by specific pharmaceuticals, e.g., the anti-overactive bladder drug propiverine or a norepinephrine/serotonin reuptake inhibitor (NSRI), resulting in massive cytosolic and nuclear accumulations in rat kidney. To assess the underlying molecular mechanism of the latter, we aimed to characterize the nature of peroxisomal and cyto-nuclear shuttling of human and rat DAAO overexpressed in three cell lines using confocal microscopy. Indeed, interference with peroxisomal transport via deletion of the PTS1 signal or PEX5 knockdown resulted in induced nuclear DAAO localization. Having demonstrated the absence of active nuclear import and employing variably sized mCherry- and/or EYFP-fusion proteins of DAAO and catalase, we showed that peroxisomal proteins ≤134 kDa can passively diffuse into mammalian cell nuclei-thereby contradicting the often-cited 40 kDa diffusion limit. Moreover, their inherent nuclear presence and nuclear accumulation subsequent to proteasome inhibition or abrogated peroxisomal transport suggests that nuclear localization is a characteristic in the lifecycle of peroxisomal proteins. Based on this molecular trafficking analysis, we suggest that pharmaceuticals like propiverine or an NSRI may interfere with peroxisomal protein targeting and import, consequently resulting in massive nuclear protein accumulation in vivo.

Structure-function relationships in human d-amino acid oxidase variants corresponding to known SNPs.

In the brain, d-amino acid oxidase plays a key role in modulating the N-methyl-d-aspartate receptor (NMDAR) activation state, catalyzing the stereospecific degradation of the coagonist d-serine. A relationship between d-serine signaling deregulation, NMDAR dysfunction, and CNS diseases is presumed. Notably, the R199W substitution in human DAAO (hDAAO) was associated with familial amyotrophic lateral sclerosis (ALS), and further coding substitutions, i.e., R199Q and W209R, were also deposited in the single nucleotide polymorphism database. Here, we investigated the biochemical properties of these different hDAAO variants. The W209R hDAAO variant shows an improved d-serine degradation ability (higher activity and affinity for the cofactor FAD) and produces a greater decrease in cellular d/(d+l) serine ratio than the wild-type counterpart when expressed in U87 cells. The production of H2O2 as result of excessive d-serine degradation by this hDAAO variant may represent the factor affecting cell viability after stable transfection. The R199W/Q substitution in hDAAO altered the protein conformation and enzymatic activity was lost under conditions resembling the cellular ones: this resulted in an abnormal increase in cellular d-serine levels. Altogether, these results indicate that substitutions that affect hDAAO functionality directly impact on d-serine cellular levels (at least in the model cell system used). The pathological effect of the expression of the R199W hDAAO, as observed in familial ALS, originates from both protein instability and a decrease in kinetic efficiency: the increase in synaptic d-serine may be mainly responsible for the neurotoxic effect. This information is expected to drive future targeted treatments.

Characterization of human DAAO variants potentially related to an increased risk of schizophrenia.

Considering the key role of d-serine in N-methyl-d-aspartate receptor-mediated neurotransmission, it is highly relevant to define the role that enzymes play in d-serine synthesis and degradation. In particular, the details of regulation of the d-serine catabolic human enzyme d-amino acid oxidase (hDAAO) are unknown although different lines of evidence have shown it to be involved in schizophrenia susceptibility. Here we investigated the effect of three single nucleotide polymorphisms and known mutations in hDAAO, i.e., D31H, R279A, and G331V. A very low amount of soluble G331V hDAAO is produced in E. coli cells: the recombinant variant enzyme is fully active. Human U87 glioblastoma cells transiently transfected for G331V hDAAO show a low viability, a significant amount of protein aggregates, and augmented apoptosis. The recombinant D31H and R279A hDAAO variants do not show alterations in tertiary and quaternary structures, thermal stability, binding affinity for inhibitors, and the modulator pLG72, whereas the kinetic efficiency and the affinity for d-serine and for FAD were higher than for the wild-type enzyme. While these effects for the substitution at position 31 cannot be structurally explained, the R279A mutation might affect the hDAAO FAD-binding affinity by altering the "structurally ambivalent" peptide V47-L51. In agreement with the observed increased activity, expression of D31H and R279A hDAAO variants in U87 cells produces a higher decrease in cellular d/(d+l) serine ratio than the wild-type counterpart. In vivo, these substitutions could affect cellular d-serine concentration and its release at synapsis and thus might be relevant for schizophrenia susceptibility.

Reduced D-serine levels in the nucleus accumbens of cocaine-treated rats hinder the induction of NMDA receptor-dependent synaptic plasticity.

Cocaine seeking behaviour and relapse have been linked to impaired potentiation and depression at excitatory synapses in the nucleus accumbens, but the mechanism underlying this process is poorly understood. We show that, in the rat nucleus accumbens core, D-serine is the endogenous coagonist of N-methyl-D-aspartate receptors, and its presence is essential for N-methyl-D-aspartate receptor-dependent potentiation and depression of synaptic transmission. Nucleus accumbens core slices obtained from cocaine-treated rats after 1 day of abstinence presented significantly reduced D-serine concentrations, increased expression of the D-serine degrading enzyme, D-amino acid oxidase, and downregulated expression of serine racemase, the enzyme responsible for D-serine synthesis. The D-serine deficit was associated with impairment of potentiation and depression of glutamatergic synaptic transmission, which was restored by slice perfusion with exogenous D-serine. Furthermore, in vivo administration of D-serine directly into the nucleus accumbens core blocked behavioural sensitization to cocaine. These results provide evidence for a critical role of D-serine signalling in synaptic plasticity relevant to cocaine addiction.

Biochemical and cellular studies of three human 3-phosphoglycerate dehydrogenase variants responsible for pathological reduced L-serine levels.

In the brain, the non-essential amino acid L-serine is produced through the phosphorylated pathway (PP) starting from the glycolytic intermediate 3-phosphoglycerate: among the different roles played by this amino acid, it can be converted into D-serine and glycine, the two main co-agonists of NMDA receptors. In humans, the enzymes of the PP, namely phosphoglycerate dehydrogenase (hPHGDH, which catalyzes the first and rate-limiting step of this pathway), 3-phosphoserine aminotransferase, and 3-phosphoserine phosphatase are likely organized in the cytosol as a metabolic assembly (a "serinosome"). The hPHGDH deficiency is a pathological condition biochemically characterized by reduced levels of L-serine in plasma and cerebrospinal fluid and clinically identified by severe neurological impairment. Here, three single-point variants responsible for hPHGDH deficiency and Neu-Laxova syndrome have been studied. Their biochemical characterization shows that V261M, V425M, and V490M substitutions alter either the kinetic (both maximal activity and Km for 3-phosphoglycerate in the physiological direction) and the structural properties (secondary, tertiary, and quaternary structure, favoring aggregation) of hPHGDH. All the three variants have been successfully ectopically expressed in U251 cells, thus the pathological effect is not due to hindered expression level. At the cellular level, mistargeting and aggregation phenomena have been observed in cells transiently expressing the pathological protein variants, as well as a reduced L-serine cellular level. Previous studies demonstrated that the pharmacological supplementation of L-serine in hPHGDH deficiencies could ameliorate some of the related symptoms: our results now suggest the use of additional and alternative therapeutic approaches.

L-serine deficiency: on the properties of the Asn133Ser variant of human phosphoserine phosphatase.

The non-essential amino acid L-serine is involved in a number of metabolic pathways and in the brain its level is largely due to the biosynthesis from the glycolytic intermediate D-3-phosphoglycerate by the phosphorylated pathway (PP). This cytosolic pathway is made by three enzymes proposed to generate a reversible metabolon named the "serinosome". Phosphoserine phosphatase (PSP) catalyses the last and irreversible step, representing the driving force pushing L-serine synthesis. Genetic defects of the PP enzymes result in strong neurological phenotypes. Recently, we identified the homozygous missense variant [NM_004577.4: c.398A > G p.(Asn133Ser)] in the PSPH, the PSP encoding gene, in two siblings with a neurodevelopmental syndrome and a myelopathy. The recombinant Asn133Ser enzyme does not show significant alterations in protein conformation and dimeric oligomerization state, as well as in enzymatic activity and functionality of the reconstructed PP. However, the Asn133Ser variant is less stable than wild-type PSP, a feature also apparent at cellular level. Studies on patients' fibroblasts also highlight a strong decrease in the level of the enzymes of the PP, a partial nuclear and perinuclear localization of variant PSP and a stronger perinuclear aggregates formation. We propose that these alterations contribute to the formation of a dysfunctional serinosome and thus to the observed reduction of L-serine, glycine and D-serine levels (the latter playing a crucial role in modulating NMDA receptors). The characterization of patients harbouring the Asn133Ser PSP substitution allows to go deep into the molecular mechanisms related to L-serine deficit and to suggest treatments to cope with the observed amino acids alterations.

D-Serine metabolism: new insights into the modulation of D-amino acid oxidase activity.

Over the years, accumulating evidence has indicated that D-serine represents the main endogenous ligand of NMDA (N-methyl-D-aspartate) receptors. In the brain, the concentration of D-serine stored in cells is defined by the activity of two enzymes: serine racemase (responsible for both the synthesis and degradation) and D-amino acid oxidase (which catalyses D-serine degradation). The present review is focused on human D-amino acid oxidase, discussing the mechanisms involved in modulating enzyme activity and stability, with the aim to substantiate the pivotal role of D-amino acid oxidase in brain D-serine metabolism.

Glial D-serine gates NMDA receptors at excitatory synapses in prefrontal cortex.

N-methyl-D-aspartate receptors (NMDARs) subserve numerous neurophysiological and neuropathological processes in the cerebral cortex. Their activation requires the binding of glutamate and also of a coagonist. Whereas glycine and D-serine (D-ser) are candidates for such a role at central synapses, the nature of the coagonist in cerebral cortex remains unknown. We first show that the glycine-binding site of NMDARs is not saturated in acute slices preparations of medial prefrontal cortex (mPFC). Using enzymes that selectively degrade either D-ser or glycine, we demonstrate that under the present conditions, D-ser is the principle endogenous coagonist of synaptic NMDARs at mature excitatory synapses in layers V/VI of mPFC where it is essential for long-term potentiation (LTP) induction. Furthermore, blocking the activity of glia with the metabolic inhibitor, fluoroacetate, impairs NMDAR-mediated synaptic transmission and prevents LTP induction by reducing the extracellular levels of D-serine. Such deficits can be restored by exogenous D-ser, indicating that the D-amino acid mainly originates from glia in the mPFC, as further confirmed by double-immunostaining studies for D-ser and anti-glial fibrillary acidic protein. Our findings suggest that D-ser modulates neuronal networks in the cerebral cortex by gating the activity of NMDARs and that altering its levels is relevant to the induction and potentially treatment of psychiatric and neurological disorders.

On the regulation of human D-aspartate oxidase.

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