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D-Serine is a D-amino acid that occurs at high levels in the mammalian brain and is an endogenous ligand of the "glycine site" of N-methyl D-aspartate (NMDA) 1 receptors (1-4). NMDA receptors play key roles in excitatory synaptic transmission, plasticity, and learning and memory (5). Overactivation of the NMDA receptor and the resultant influx of calcium into cells is a major culprit in the cell death that occurs following stroke and neurodegenerative diseases. Blockers of the "glycine site" of the receptor are neuroprotective in animal models of stroke (5). Endogenous D-serine is required for NMDA receptor activation, and its removal markedly decreases NMDA receptor activity (3). In the vertebrate retina, endogenous D-serine may also mediate the light-dependent increase in neuronal activity by activating NMDA receptors (6). More recently, D-serine was suggested to play a role in the long term potentiation of synaptic transmission in the hippocampus, indicating a role of endogenous D-serine in long term synaptic plasticity (7).D-Serine is synthesized by serine racemase, a pyridoxal phosphate (PLP)-dependent enzyme enriched in the mammalian brain (8, 9). Serine racemase has high sequence homology with the fold-type II group of PLP enzymes, such as serine/threonine dehydratase and D-serine dehydratase (10, 11). In addition to converting L-to D-serine, serine racemase catalyzes the ␣,␤-elimination of water from L-serine to form pyruvate and ammonia (12). The initial rates of racemization and ␣,␤-elimination of L-serine by serine racemase are strongly stimulated by magnesium and ATP, indicating that the complex Mg⅐ATP is a physiological ligand of the enzyme (12).In accordance with accepted mechanisms of PLP-catalyzed reactions (13-16), a mechanism for racemization and ␣,␤-elimination catalyzed by serine racemase is depicted in Scheme 1. PLP, bound to the enzyme through an internal aldimine with The termination of signaling by a neurotransmitter in the brain normally requires its re-uptake and metabolism. D-Serine signaling is thought to involve its release from cells to

N-methyl d-aspartate receptors (NMDARs) are key excitatory neurotransmitter receptors in the brain and are involved in many physiological processes, including memory formation, synaptic plasticity and development [1]. The NMDARs are composed of multiple subunits and their activity is regulated by numerous mechanisms, including different ligands and interacting proteins [2]. The NMDARs display high permeability to Ca 2+ , which is known to play a central role in syn-aptic plasticity and many signal transduction mechanisms [1]. NMDAR overstimulation promotes neurotoxicity and is implicated in several pathological conditions, such as stroke and neurodegenerative diseases [3]. The NMDARs are unique in their requirement for more than one agonist to operate. Glutamate, the main NMDAR agonist, does not activate the receptors unless a co-agonist binding site located at the NR1 subunit is occupied [4,5]. d-Serine, an unusual d-amino acid present in mammalian brain, is now recognized as a physiological ligand of the NMDAR co-agonist site, mediating several NMDAR-dependent processes [6-15]. At first, the NMDAR co-agonist site was thought to be occupied by glycine. Hence, the co-agonist site is also generally referred to as the 'glycine site'. In addition, to be essential for NMDAR activity, the co-agonist site exerts neuromodulatory roles. Thus, co-agonist binding increases the receptor's affinity for glutamate [16], decreases its desensitization [17] and promotes NMDAR turnover by internalization [18]. Since its discovery, the role of the co-agonist site in regulating the activity of the NMDAR has been 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, degrada-tive 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. Abbreviations ALS, amyotrophic lateral sclerosis; AMPA, a-amino-3-hydroxy-5-methylisoxazole-4-propionic a...

High levels of D-serine occur in the brain, challenging the notion that D-amino acids would not be present or play a role in mammals. D-serine levels in the brain are even higher than many L-amino acids, such as asparagine, valine, isoleucine, and tryptophan, among others. D-serine is synthesized by a serine racemase (SR) enzyme, which directly converts L-to D-serine. We now report that SR is a bifunctional enzyme, producing both D-serine and pyruvate in cultured cells and in vitro. Transfection of SR into HEK 293 cells elicits synthesis of D-serine and augmented release of pyruvate to culture media. We identified substances present in HEK 293 and astrocyte cell extracts that strongly stimulate D-serine production by SR and elicit production of pyruvate. Experiments with recombinant enzyme reveal that Mg 2؉ and ATP present in the cell extracts are physiological cofactors and increase 5-to 10-fold the rates of racemization and production of pyruvate. As much as three molecules of pyruvate are synthesized for each molecule of D-serine produced by SR. This finding constitutes a previously undescribed mechanism underlying D-amino acid synthesis in mammals, different from classical amino acid racemases present in bacteria. Our data link the production of D-serine to the energy metabolism, with implications for the metabolic and transmitter crosstalk between glia and neurons.glutamate receptors ͉ pyruvate ͉ astrocytes I n recent years, it has been shown that astrocytes and neurons exhibit a dynamic bidirectional signaling that profoundly influences neuronal activity and development (1). Astrocytes modulate synaptic transmission by releasing chemical transmitters and eliciting Ca 2ϩ waves in nearby neurons. The search for new transmitter molecules in the brain uncovered the presence of high levels of D-serine in astrocytes, a D-amino acid not previously thought to occur in mammals (2, 3). D-serine is synthesized by a glial-enriched enzyme serine racemase (SR), which directly converts L-to D-serine (4-7). SR does not bear significant homology with bacterial racemases, and little is known about the mechanism and regulation of D-serine production.D-serine released from astrocytes seems to be an endogenous ligand of the N-methyl-D-aspartate (NMDA) receptor (3,8). Depletion of endogenous D-serine in slices and cultured cells strongly diminishes NMDA receptor responses measured biochemically and electrophysiologically (8). Massive stimulation of NMDA receptors is implicated in neural damage after stroke (9), and inhibitors of SR may be useful to prevent stroke damage. Thus, inhibitors of SR provide a strategy to decrease NMDA receptor coactivation and may be useful in conditions in which overstimulation of NMDA receptors plays a pathological role.To clarify the role of D-serine as a modulator of NMDA receptors, one should identify the factors that regulate SR and D-serine signaling. In this paper, we explored mechanisms regulating the production of D-serine by SR. We demonstrate that SR is a unique bifunctional enzyme, produ...

Astrocytes express the 3-phosphoglycerate dehydrogenase (Phgdh) enzyme required for the synthesis of l-serine from glucose. Astrocytic l-serine was proposed to regulate NMDAR activity by shuttling to neurons to sustain d-serine production, but this hypothesis remains untested. We now report that inhibition of astrocytic Phgdh suppressed the de novo synthesis of l-and d-serine and reduced the NMDAR synaptic potentials and long-term potentiation (LTP) at the Schaffer collaterals-CA1 synapse. Likewise, enzymatic removal of extracellular l-serine impaired LTP, supporting an l-serine shuttle mechanism between glia and neurons in generating the NMDAR coagonist d-serine. Moreover, deletion of serine racemase (SR) in glutamatergic neurons abrogated d-serine synthesis to the same extent as Phgdh inhibition, suggesting that neurons are the predominant source of the newly synthesized d-serine. We also found that the synaptic NMDAR activation in adult SR-knockout (KO) mice requires Phgdh-derived glycine, despite the sharp decline in the postnatal glycine levels as a result of the emergence of the glycine cleavage system. Unexpectedly, we also discovered that glycine regulates d-serine metabolism by a dual mechanism. The first consists of tonic inhibition of SR by intracellular glycine observed in vitro, primary cultures, and in vivo microdialysis. The second involves a transient glycine-induce d-serine release through the Asc-1 transporter, an effect abolished in Asc-1 KO mice and diminished by deleting SR in glutamatergic neurons. Our observations suggest that glycine is a multifaceted regulator of d-serine metabolism and implicate both d-serine and glycine in mediating NMDAR synaptic activation at the mature hippocampus through a Phgdh-dependent shuttle mechanism.

d-Serine is a co-agonist of NMDA receptors (NMDARs) whose activity is potentially regulated by Asc-1 (SLC7A10), a transporter that displays high affinity for d-serine and glycine. Asc-1 operates as a facilitative transporter and as an antiporter, though the preferred direction of d-serine transport is uncertain. We developed a selective Asc-1 blocker, Lu AE00527, that blocks d-serine release mediated by all the transport modes of Asc-1 in primary cultures and neocortical slices. Furthermore, d-serine release is reduced in slices from Asc-1 knockout (KO) mice, indicating that d-serine efflux is the preferred direction of Asc-1. The selectivity of Lu AE00527 is assured by the lack of effect on slices from Asc-1-KO mice, and the lack of interaction with the co-agonist site of NMDARs. Moreover, in vivo injection of Lu AE00527 in P-glycoprotein-deficient mice recapitulates a hyperekplexia-like phenotype similar to that in Asc-1-KO mice. In slices, Lu AE00527 decreases the long-term potentiation at the Schaffer collateral-CA1 synapses, but does not affect the long-term depression. Lu AE00527 blocks NMDAR synaptic potentials when typical Asc-1 extracellular substrates are present, but it does not affect AMPAR transmission. Our data demonstrate that Asc-1 mediates tonic co-agonist release, which is required for optimal NMDAR activation and synaptic plasticity.

D-serine is a physiological coagonist of N-methyl D-aspartate receptors (NMDARs) that plays a major role in several NMDARdependent events. In this study we investigate mechanisms regulating D-serine production by the enzyme serine racemase (SR). We now report that NMDAR activation promotes translocation of SR to the plasma membrane, which dramatically reduces the enzyme activity. Membrane-bound SR isolated from rat brain is not extracted from the membrane by high detergent and salt concentration, indicating a strong association. Colocalization studies indicate that most membrane-bound SR is located at the plasma membrane and dendrites, with much less SR observed in other types of membrane. NMDAR activation promotes translocation of the cytosolic SR to the membrane, resulting in reduced D-serine synthesis, and this effect is averted by blockade of NMDARs. In primary neuronal cultures, SR translocation to the membrane is blocked by a palmitoylation inhibitor, indicating that membrane binding is mediated by fatty acid acylation of SR. In agreement, we found that SR is acylated in transfected neuroblastoma cells using [ 3 H]palmitate or [ 3 H]octanoic acid as precursors. In contrast to classical S-palmitoylation of cysteines, acylation of SR occurs through the formation of an oxyester bond with serine or threonine residues. In addition, we show that phosphorylation of Thr-227 is also required for steady-state binding of SR to the membrane under basal, nonstimulated condition. We propose that the inhibition of D-serine synthesis caused by translocation of SR to the membrane provides a fail-safe mechanism to prevent NMDAR overactivation in vicinal cells or synapses.glutamate ͉ neurotransmission ͉ octanoylation ͉ palmitoylation ͉ synapse D -serine is a physiological ligand of the coagonist site of NMDARs, mediating several NMDAR-dependent events, including NMDAR neurotransmission (1), neurotoxicity (2, 3), synaptic plasticity (4), and cell migration (5). D-serine is synthesized by serine racemase (SR), an enzyme that directly converts L-into D-serine (6). This enzyme is regulated by interacting proteins, such as the glutamate interacting protein 1 (5), Pick-1 (7), and Golga3 (8), and by nitric oxide produced upon NMDAR activation (9).Despite the many roles attributed to it, the regulation of D-serine signaling is still largely unknown. Furthermore, many questions remain unresolved regarding the distribution of SR and the roles played by glia vs. neurons in D-serine signaling (10). Although the highest levels of endogenous D-serine were shown to be present in brain astrocytes (11), D-serine has also been detected in neurons (2). Recent data using new antibodies against SR (2) and SR knockout mice as negative controls (12) indicate that SR is abundantly expressed in neurons, with highest levels in the cerebral cortex and the hippocampal formation. Moreover, endogenous D-serine released from neuronal cultures lacking significant levels of astrocytes mediates NMDAR-elicited neurotoxicity (2), suggesting that neuron-derived...

Mammalian serine racemase is a brain-enriched enzyme that converts L-into D-serine in the nervous system. D-Serine is an endogenous co-agonist at the "glycine site" of N-methyl D-aspartate (NMDA) receptors that is required for the receptor/ channel opening. Factors regulating the synthesis of D-serine have implications for the NMDA receptor transmission, but little is known on the signals and events affecting serine racemase levels. We found that serine racemase interacts with the Golgin subfamily A member 3 (Golga3) protein in yeast two-hybrid screening. The interaction was confirmed in vitro with the recombinant proteins in co-transfected HEK293 cells and in vivo by co-immunoprecipitation studies from brain homogenates. Golga3 and serine racemase co-localized at the cytosol, perinuclear Golgi region, and neuronal and glial cell processes in primary cultures. Golga3 significantly increased serine racemase steady-state levels in co-transfected HEK293 cells and primary astrocyte cultures. This observation led us to investigate mechanisms regulating serine racemase levels. We found that serine racemase is degraded through the ubiquitin-proteasomal system in a Golga3-modulated manner. Golga3 decreased the ubiquitylation of serine racemase both in vitro and in vivo and significantly increased the protein half-life in pulse-chase experiments. Our results suggest that the ubiquitin system is a main regulator of serine racemase and D-serine levels. Modulation of serine racemase degradation, such as that promoted by Golga3, provides a new mechanism for regulating brain D-serine levels and NMDA receptor activity.2 type of glutamate receptors play key roles in excitatory synaptic transmission and are involved in many physiological processes including learning and memory (1). NMDA receptor activity is tightly regulated, as its overactivation contributes to pathologic conditions such as stroke and neurodegenerative diseases (2). An interesting feature of NMDA receptors is the requirement of simultaneous binding of two agonists for channel opening, i.e. the NMDA channel only operates when both a glutamate site and a coagonist site are occupied (1). It has been shown that binding of glycine to the co-agonist site is an obligatory requirement for NMDA receptor/channel operation (3, 4). Subsequent studies have shown that brain D-serine is an endogenous ligand of the glycine site of NMDA receptors (5-9).Regulation of NMDA receptor activity by the co-agonist D-serine plays critical roles. Removal of endogenous D-serine decreases NMDA receptor responses (8) and blocks NMDAdependent migration of immature granule cells in the cerebellum (9). D-Serine is the dominant endogenous co-agonist for NMDA neurotoxicity, as removal of D-serine abolishes NMDA receptor-elicited cell death in hippocampal slices (6). In the vertebrate retina, endogenous D-serine mediates the light-dependent increase in neuronal activity by activating NMDA receptors (10). Furthermore, endogenous D-serine is required for the long term potentiation of the synaptic t...