Protein kinase C activity regulates D-serine availability in the brain

D-serine is a co-agonist of NMDA receptor (NMDAR) and plays important roles in synaptic plasticity mechanisms. Serine racemase (SR) is a brain-enriched enzyme that converts L-serine to D-serine. SR interacts with the protein interacting with C-kinase 1 (PICK1), which is known to direct protein kinase C (PKC) to its targets in cells. Here, we investigated whether PKC activity regulates SR activity and D-serine availability in the brain. In vitro, PKC phosphorylated SR and decreased its activity. PKC activation increased SR phosphorylation in serine residues and reduced D-serine levels in astrocyte and neuronal cultures. Conversely, PKC inhibition decreased basal SR phosphorylation and increased cellular D-serine levels. In vivo modulation of PKC activity regulated both SR phosphorylation and D-serine levels in rat frontal cortex. Finally, rats that completed an object recognition task showed decreased SR phosphorylation and increased D-serine/total serine ratios, which was markedly correlated with decreased PKC activity in both cortex and hippocampus. Results indicate that PKC phosphorylates SR in serine residues and regulates D-serine availability in the brain. This interaction may be relevant for the regulation of physiological and pathological mechanisms linked to NMDAR function.     

AMPA receptor mediated D-serine release from retinal glial cells

The N-methyl-D-aspartate receptor (NMDAR) co-agonist D-serine is important in a number of different processes in the CNS, ranging from synaptic plasticity to disease states, including schizophrenia. D-serine appears to be the major co-agonist acting on retinal ganglion cell NMDA receptors, but the cell type from which it originates and whether its release can be modulated by activity are unknown. In this study, we utilized a mutant mouse line with elevated d-serine to investigate this question. Direct measurements of extracellular D-serine using capillary electrophoresis demonstrate that D-serine can be released from the intact mouse retina through an α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate receptor (AMPAR) dependent mechanism. α-Amino-3-hydroxyl-5-methyl-4-isoxazole-propionate-evoked D-serine release persisted in the presence of a cocktail of neural inhibitors but was abolished after administration of a glial toxin. These findings provide the first evidence that extracellular D-serine levels in the retina can be modulated, and that such modulation is contingent upon glial cell activity.     

Paradoxical roles of serine racemase and D-serine in the G93A mSOD1 mouse model of amyotrophic lateral sclerosis

D-serine is an endogenous neurotransmitter that binds to the NMDA receptor, thereby increasing the affinity for glutamate, and the potential for excitotoxicity. The primary source of D-serine in vivo is enzymatic racemization by serine racemase (SR). Regulation of D-serine in vivo is poorly understood, but is thought to involve a combination of controlled production, synaptic reuptake by transporters, and intracellular degradation by D-amino acid oxidase (DAO). However, SR itself possesses a well-characterized eliminase activity, which effectively degrades D-serine as well. D-serine is increased two-fold in spinal cords of G93A Cu,Zn-superoxide dismutase (SOD1) mice--the standard model of amyotrophic lateral sclerosis (ALS). ALS mice with SR disruption show earlier symptom onset, but survive longer (progression phase is slowed), in an SR-dependent manner. Paradoxically, administration of D-serine to ALS mice dramatically lowers cord levels of D-serine, leading to changes in the onset and survival very similar to SR deletion. D-serine treatment also increases cord levels of the alanine-serine-cysteine transporter 1 (Asc-1). Although the mechanism by which SOD1 mutations increases D-serine is not known, these results strongly suggest that SR and D-serine are fundamentally involved in both the pre-symptomatic and progression phases of disease, and offer a direct link between mutant SOD1 and a glial-derived toxic mediator.     

Osteoclastogenesis is negatively regulated by D-serine produced by osteoblasts

We have shown the functional expression by chondrocytes of serine racemase (SR) which is responsible for the synthesis of D-serine (Ser) from L-Ser in cartilage. In this study, we evaluated the possible functional expression of SR by bone-forming osteoblasts and bone-resorbing osteoclasts. Expression of SR mRNA was seen in osteoblasts localized at the cancellous bone surface in neonatal rat tibial sections and in cultured rat calvarial osteoblasts endowed to release D-Ser into extracellular medium, but not in cultured osteoclasts differentiated from murine bone marrow progenitor cells. Sustained exposure to D-Ser failed to significantly affect alkaline phosphatase activity and Ca(2+) accumulation in cultured osteoblasts, but significantly inhibited differentiation and maturation in a concentration-dependent manner at a concentration range of 0.1-1 mM without affecting cellular survival in cultured osteoclasts. By contrast, L-Ser promoted osteoclastic differentiation in a manner sensitive to the inhibition by D-Ser. Matured osteoclasts expressed mRNA for the amino acid transporter B(0,+) (ATB(0,+) ) and the system alanine, serine, and cysteine amino acid transporter-2 (ASCT2), which are individually capable of similarly incorporating extracellular L- and D-Ser. Knockdown of these transporters by siRNA prevented both the promotion by L-Ser and the inhibition by D-Ser of osteoclastic differentiation in pre-osteoclastic RAW264.7 cells. These results suggest that D-Ser may play a pivotal role in osteoclastogenesis through a mechanism related to the incorporation mediated by both ATB(0,+) and ASCT2 of serine enantiomers in osteoclasts after the synthesis and subsequent release from adjacent osteoblasts.     

Potential role for astroglial D-amino acid oxidase in extracellular D-serine metabolism and cytotoxicity

D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of D-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, D-serine is proposed to be a neuromodulator of the N-methyl-D-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular D-serine in glial cells. Here we show that after D-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of D-serine led to the production of hydrogen peroxide (H(2)O(2)) followed by apoptosis in C6/DAO cells. Among the amino acids tested, D-serine specifically exhibited a significant cell death-inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with D-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular D-serine can gain access to intracellular DAO, being metabolized to produce H(2)O(2). These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, D-serine.     

Molecular characterization of three forms of vitellogenin and their yolk protein products during oocyte growth and maturation in red seabream (Pagrus major), a marine teleost spawning pelagic eggs

Full-length cDNAs encoding three forms of vitellogenin (Vg) were obtained from a liver cDNA library of estrogen-treated red seabream, Pagrus major. Two of the three Vg sequences had high homology with type-A and -B Vgs (VgA and VgB) of other teleosts. The third red seabream Vg was classified as a type-C or phosvitinless (Pvl) Vg due to its lack of a phosvitin (Pv) domain. Two Vg preparations (610 and 340 kDa) from blood serum of estradiol-treated fish were biochemically characterized. Analyses of precursor-product relationships by examination of N-terminal amino acid sequences verified cleavage of the 610 kDa Vg into a 540 kDa lipovitellin (Lv) and a 32 kDa beta'-component. Each of these yolk preparations comprising both VgA- and VgB-derived polypeptides. The 340 kDa Vg, which was immunologically verified to be a PvlVg, was accumulated by vitellogenic oocytes with no alterations to its native molecular mass. During oocyte maturation, the VgA- and VgB-derived yolk proteins were differentially processed, presumably to generate a pool of free amino acids for oocyte hydration or for allocation of specific types of nutrients, amino acids, and proteins, to the developing embryo. Conversely, the 340 kDa Vg-derived yolk protein is unlikely to contribute to oocyte hydration or diffusible nutrients since the molecule underwent only minor proteolytic nicking during oogenesis. The present study elucidates for the first time specific functions of three different forms of Vg and their product yolk proteins in a higher taxonomic group of marine teleosts that spawn pelagic eggs.     

D构象丝氨酸在中枢神经系统中的功能研究进展

哺乳动物中枢神经系统中D构象丝氨酸的区域性高浓度分布与N-甲基-D-天冬氨酸(NMDA)受体相一致.它主要由丝氨酸消旋酶将L丝氨酸直接消旋而来,也可能通过肠道菌群产生后吸收至体内,最终被D构象氨基酸氧化酶氧化.这种从胶质细胞而非神经元来源的“异常”构象氨基酸作为一种新型神经递质,不仅更新了传统“神经递质”的定义,而且为许多与NMDA受体过度兴奋或表达下调相关的神经系统疾病治疗提出了新的线索.     

DNA sequence of the D-serine deaminase activator gene dsdC.

We have determined the DNA sequence of dsdC, the gene that encodes the D-serine deaminase activator protein of Escherichia coli K-12. The sequence contains a single open reading frame that terminates in a UGA codon. One the basis of the size of the protein, 33 kilodaltons, and the amino acid sequence encoded by the open reading frame, we identified a likely translation initiation codon 731 base pairs upstream of the translation initiation codon for the divergently transcribed D-serine deaminase gene. There is a broad range of codon usage, not surprising in view of the weak expression of the gene. The N-terminal two-thirds of the activator is arginine-lysine rich and quite polar; the remainder is more neutral. The segment of the protein that seems most likely to have potential to form the helix-turn-helix structure characteristic of DNA-regulatory proteins is located near the end of the polar region. The protein contains a region with significant homology to lambda attB.     

D-amino acids in the brain: D-serine in neurotransmission and neurodegeneration

The mammalian brain contains unusually high levels of D-serine, a D-amino acid previously thought to be restricted to some bacteria and insects. In the last few years, studies from several groups have demonstrated that D-serine is a physiological co-agonist of the N-methyl D-aspartate (NMDA) type of glutamate receptor -- a key excitatory neurotransmitter receptor in the brain. D-Serine binds with high affinity to a co-agonist site at the NMDA receptors and, along with glutamate, mediates several important physiological and pathological processes, including NMDA receptor transmission, synaptic plasticity and neurotoxicity. In recent years, biosynthetic, degradative and release pathways for D-serine have been identified, indicating that D-serine may function as a transmitter. At first, D-serine was described in astrocytes, a class of glial cells that ensheathes neurons and release several transmitters that modulate neurotransmission. This led to the notion that D-serine is a glia-derived transmitter (or gliotransmitter). However, recent data indicate that serine racemase, the D-serine biosynthetic enzyme, is widely expressed in neurons of the brain, suggesting that D-serine also has a neuronal origin. We now review these findings, focusing on recent questions regarding the roles of glia versus neurons in d-serine signaling.     

Analysis of free D-serine in mammals and its biological relevance

D-Serine is a unique endogenous substance enriched in the brain at the exceptionally high concentrations as a free D-amino acid in mammals throughout their life. Peripheral tissues and blood contain low or trace levels of the D-amino acid. In the nervous systems, D-serine appears to act as an intrinsic coagonist for the N-methyl-D-aspartate type glutamate receptor (NMDA receptor) based upon the following characteristics: (i) D-serine stereoselectively binds to and stimulates the glycine-regulatory site of the NMDA receptor consisting of GRIN1/GRIN2 subunits more potently than glycine with an affinity and ED50 at high nanomolar ranges, (ii) the selective elimination of D-serine in brain tissues attenuates the NMDA receptor functions, indicating an indispensable role in physiological activation of the glutamate receptor, and (iii) the distribution of D-serine is uneven and closely correlated with that of the binding densities of the various NMDA receptor sites, and especially of the GRIN2B subunit in the brain. Moreover, d-serine exerts substantial influence on the GRIN1/GRIN3-NMDA and δ2 glutamate receptor. In the brain and retina, metabolic processes of D-serine, such as biosynthesis, extracellular release, uptake, and degradation, are observed and some candidate molecules that mediate these processes have been isolated. The fact that the mode of extracellular release of D-serine in the brain differs from that of classical neurotransmitters is likely to be related to the detection of D-serine in both glial cells and neurons, suggesting that d-serine signals could be required for the glia-synapse interaction. Moreover, the findings from the basic experiments and clinical observations support the views that the signaling system of endogenous free D-serine plays important roles, at least, through the action on the NMDA receptors in the brain wiring development and the regulation of higher brain functions, including cognitive, emotional and sensorimotor function. Based upon these data, aberrant D-serine-NMDA receptor interactions have been considered to be involved in the pathophysiology of a variety of neuropsychiatric disorders including schizophrenia and ischemic neuronal cell death. The molecular and cellular mechanisms for regulating the D-serine signals in the nervous system are, therefore, suitable targets for studies aiming to elucidate the causes of neuropsychiatric disorders and for the development of new treatments for intractable neuropsychiatric symptoms.     

Rapid determination of free D-serine with chicken D-serine dehydratase

We have developed a simple, rapid, and inexpensive method of measuring the concentration of intrinsic free D-serine in tissue samples. This method uses chicken D-serine dehydratase in an enzymatic reaction to produce pyruvate, which is detected spectrophotometrically. Pyridoxal 5'-phosphate (PLP), a cofactor of D-serine dehydratase, increased pyruvate formation by 28%. The presence of Zn(2+) or ethylenediaminetetraacetic acid (EDTA) did not have any effect on pyruvate formation under the present assay conditions. In addition, this method was not affected by the presence of a large excess of L-serine, nor by the presence of tissue extracts, and accurately determined concentrations of 2-30 μM (200 pmol-3 nmol) of D-serine. The entire assay requires only 60 min. With this method, we determined the concentration of D-serine in various silkworm tissues. The results were in agreement with high performance liquid chromatography measurements. We found high concentrations of D-serine in silkworm larvae at day 3 of the fifth instar; specifically, 509 nmol g(-1) wet tissue in the midgut, 434 nmol g(-1) in the ovary, and 353 nmol g(-1) in the testis.     

Neuron-derived D-serine release provides a novel means to activate N-methyl-D-aspartate receptors

D-serine is a coagonist of N-methyl-D-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of D-serine is carried out by serine racemase, which converts L- to D-serine. D-serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of D-serine. We now report significant amounts of serine racemase and D-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and D-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and D-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived D-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous D-serine by addition of the recombinant enzyme D-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal D-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked D-serine release. Release of D-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. D-serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of D-serine. Glutamate-induced neuronal D-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way.     

Identification and Characterization of the Human Ortholog of Rat STXBP1, a Protein Implicated in Vesicle Trafficking and Neurotransmitter Release

In a screen designed to identify genes expressed preferentially in retina, we identified a cDNA encoding the human ortholog of rat STXBP1 (n-Sec1, Munc-18-1, rbSec1), a protein implicated in vesicle trafficking and neurotransmitter release. This protein also has similarity toDrosophilaRop (64% aa identity) andCaenorhabditis elegansUNC-18 (58% aa identity). The major human cDNA encodes a protein of 594 amino acids which has 100% amino acid identity with its rat and murine counterparts. Additionally, there is an alternative splice form in humans, arising from the inclusion of an additional exon, which encodes a protein of 603 amino acids and is also 100% identical to the corresponding rat isoform. We found expression of the shorter cDNA in all tissues and cell lines we examined with highest levels in retina and cerebellum. By RT-PCR analysis, we found expression of the longer cDNA in neural tissues only. We mapped the structural gene to 9q34.1, a region without obvious candidate phenotypes. However, due to its evolutionary conservation and abundant expression in retina and brain, STXBP1 should be considered a candidate gene for retinal and/or neural disorders mapping to 9q34.1.     

Transport of D-serine via the amino acid transporter ATB(0,+) expressed in the colon

D-Serine, synthesized endogenously in the brain, is an important modulator of glutamatergic neurotransmission. Since colonic bacteria produce D-serine, we asked the question whether there are transport mechanisms in the colon that might make this exogenously produced D-serine available to the host. Here we identify for the first time an amino acid transporter in the intestine for high-affinity active transport of D-serine. This transporter, called ATB(0,+), is a Na(+)- and Cl(-)-coupled transporter for L-enantiomers of neutral and cationic amino acids. Here we demonstrate that ATB(0,+) is also capable of mediating the Na(+)- and Cl(-)-coupled transport of D-serine. The affinity of ATB(0,+) for L-serine and D-serine is similar, the K(t) value for the two enantiomers being approximately 150 microM. In addition to D-serine, ATB(0,+) transports D-alanine, D-methionine, D-leucine, and D-tryptophan. However, several other neutral and cationic amino acids that are transportable substrates for ATB(0,+) as L-enantiomers are not transported when presented as D-enantiomers. ATB(0,+) is expressed in the intestinal tract, interestingly not in the proximal intestine but in the distal intestine. Expression is most predominant in the colon where the transporter is localized to the luminal membrane of colonocytes, making this transporter uniquely suitable for absorption of bacteria-derived D-serine.     

Brain D-serine and tyrosine levels in ataxic mutant mice

Summary Since D-serine occurs at high concentrations in mammalian forebrains, the brain D-serine content was analyzed in hyperkinetic and ataxic mutant mice as well as normal control mice in a search for a physiological role. The concentrations of free D-serine (nmol/g wet weight) were 392 ± 114 (mean ± S.D.), 43 ± 17 and 18 ± 8.4 in the cerebrum, brain stem and cerebellum of the BUS mouse, respectively; and 336 ± 93,58 ± 11 and 18 ± 8.5 in the cerebrum, brain stem and cerebellum of the Rolling mouse, respectively. These values were not significantly different from those for each control animal. The present results suggest that brain D-serine may not be a cause of the abnormal movements of the mutant mice. On the contrary, among many amino acids examined, tyrosine level was found to be lower in the brain stem of BUS mouse compared to the normal control animal by amino acid analysis.     

The primary structure of the alpha subunit of a starfish guanosine-nucleotide-binding regulatory protein involved in 1-methyladenine-induced oocyte maturation.

Starfish-oocyte maturation induced by 1-methyladenine (MeAde) was inhibited by microinjection of pertussis toxin (PTX). The inhibition appeared to result from PTX-catalyzed ADP-ribosylation of a 39-kDa guanosine-nucleotide-binding regulatory protein (G protein) in the oocyte. These results strongly support the hypothesis that the MeAde-induced signals operate via a membrane receptor and are carried by the PTX-sensitive G protein. When PTX-injected oocytes were treated with dithiothreitol, 85% of them reinitiated meiosis, suggesting that dithiothreitol did not act on the MeAde receptor. We constructed a cDNA library from the immature ovary of starfish, Asterina pectinifera, and screened it with the cDNA of the alpha subunit of an inhibitory rat G protein (Gi-2). A positive cDNA clone contained an open reading frame of 1062 bases which had 74% identity with the rat Gi-2 cDNA. The deduced amino acid sequence was 85% and 89% identical to rat Gi-2 and rat Gi-1, respectively. The alpha subunit of the G protein purified from cortices of starfish oocytes was digested by trypsin and the resulting four peptides were microsequenced. Comparison of these amino acid sequences with the predicted one indicated that the isolated cDNA clone encoded the alpha subunit of the PTX-sensitive G protein in oocytes. The C-terminal sequence, KNNLKDCGLF, was identical to that of Gi, suggesting that the cysteine residue is the site of ADP-ribosylation.     

Transport systems of serine at the brain barriers and in brain parenchymal cells

D-Serine is a co-agonist for NMDA-type glutamate receptors. Although D-serine levels in CSF and interstitial fluid (ISF) affect CNS function, the regulatory system remains to be fully understood. Therefore, the purpose of this study was to investigate d-serine transport across the blood-brain barrier (BBB) and blood-CSF barrier (BCSFB) and in brain parenchymal cells. D-Serine microinjected into the cerebrum was not eliminated, suggesting a negligible contribution of D-serine efflux transport at the BBB. In contrast, D-serine was taken up from the circulating blood across the BBB via a carrier-mediated process. D-Serine elimination clearance from CSF was fourfold greater than that of d-mannitol, which is considered to reflect CSF bulk flow. The characteristics of D-serine uptake by isolated choroid plexus were consistent with those of Na(+)-independent alanine-serine-cysteine transporter 1 (asc-1). Uptake of D-serine by brain slices appeared to occur predominantly via asc-1 and Na(+)-dependent alanine-serine-cysteine transporter 2. These findings suggest that the regulatory system of D-serine levels in ISF and CSF involves (i) asc-1 at the BCSFB, acting as a major pathway of D-serine elimination from the CSF, (ii) blood-to-brain and blood-to-CSF influx transport of D-serine across the BBB and BCSFB, and (iii) concentrative uptake of D-serine by brain parenchymal cells.