The N-methyl-D-aspartate (NMDA) 2 type of glutamate receptor plays prominent roles in excitatory neurotransmission (1). In addition to glutamate, the NMDA receptor requires the obligatory binding of a coagonist of the NR1 subunit to mediate ion influx (2). Although glycine was originally suggested as the NMDA receptor coagonist, recent data indicate that endogenous D-serine is a physiologically relevant NMDA receptor ligand at the coagonist site. D-Serine is present at high levels in the brain, with little levels in peripheral tissues (3-5). Destruction of endogenous D-serine by D-amino-acid oxidase in hippocampal cultures promotes a decrease in NMDA receptor responses (6). Likewise, NMDA receptor-mediated responses in the retina and induction of long-term hippocampal potentiation are also diminished by removing D-serine (7,8). Very recently, D-serine was shown to be required for granule cell migration in the developing cerebellum (9). It has been proposed that Bergman glial cells release D-serine and enhance cell migration through activation of NMDA receptors in migrating granule cells (9). In hippocampal organotypic slice cultures, endogenous D-serine was shown to be the dominant coagonist for NMDA receptor-elicited neurotoxicity, mediating virtually all cell death elicited by NMDA (10).D-Serine is synthesized from L-serine by serine racemase, a brain enriched enzyme (11-13). Serine racemase activity is stimulated by ATP, and the enzyme also catalyzes deamination of serine into pyruvate and ammonia (14, 15). Both D-serine and serine racemase were shown previously to be enriched in astrocytes (13, 16). Purified astrocytic cultures release D-serine following (Ϯ)-␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)/kainate receptor activation (16 -18) and through an amino acid exchange mechanism by the neutral amino acid transporter ASCT (17). It has been proposed that D-serine released from astrocytes that ensheath the synapse will stimulate nearby neuronal NMDA receptors (16).The evidence that D-serine possesses a target receptor as well as a biosynthetic and degradative apparatus implies that D-serine is an important transmitter/neuromodulator in the brain (5, 19). On the other hand, the glial localization of D-serine is not compatible with the classical definition of neurotransmitter, which should be present in neurons. We have explored a possible neuronal localization of D-serine and serine racemase utilizing biochemical methods in cell cultures and new antibodies against D-serine and serine racemase. We demonstrate the unambiguous presence of serine racemase and D-serine in neurons. Neuronal release of D-serine mediates a significant fraction of NMDAelicited excitotoxicity in cortical cultures. Our data indicate that neuronal D-serine may play an important role in NMDA receptor activation, such as occurs in neurotoxicity. EXPERIMENTAL PROCEDURESMaterials-L-Serine and D-serine were purchased from Bachem. Acetonitrile, AMPA, trans-(Ϯ)-1-amino-1,3-cyclopentanedicarboxylic acid, bafilomycin A 1 , DNase I...

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...

Accurate segregation of chromosomes in mitosis is ensured by a surveillance mechanism called the mitotic (or spindle assembly) checkpoint. It prevents sister chromatid separation until all chromosomes are correctly attached to the mitotic spindle through their kinetochores. The checkpoint acts by inhibiting the anaphase-promoting complex/cyclosome (APC/C), a ubiquitin ligase that targets for degradation securin, an inhibitor of anaphase initiation. The activity of APC/C is inhibited by a mitotic checkpoint complex (MCC), composed of the APC/C activator Cdc20 bound to the checkpoint proteins MAD2, BubR1, and Bub3. When all kinetochores acquire bipolar attachment the checkpoint is inactivated, but the mechanisms of checkpoint inactivation are not understood. We have previously observed that hydrolyzable ATP is required for exit from checkpoint-arrested state. In this investigation we examined the possibility that ATP hydrolysis in exit from checkpoint is linked to the action of the Mad2-binding protein p31 comet in this process. It is known that p31 comet prevents the formation of a Mad2 dimer that it thought to be important for turning on the mitotic checkpoint. This explains how p31 comet blocks the activation of the checkpoint but not how it promotes its inactivation. Using extracts from checkpoint-arrested cells and MCC isolated from such extracts, we now show that p31 comet causes the disassembly of MCC and that this process requires β,γ-hydrolyzable ATP. Although p31 comet binds to Mad2, it promotes the dissociation of Cdc20 from BubR1 in MCC.

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...