Functional activity of N-methyl-D-aspartate (NMDA) receptors requires both glutamate binding and the binding of an endogenous coagonist that has been presumed to be glycine, although D-serine is a more potent agonist. Localizations of D-serine and it biosynthetic enzyme serine racemase approximate the distribution of NMDA receptors more closely than glycine. We now show that selective degradation of D-serine with D-amino acid oxidase greatly attenuates NMDA receptor-mediated neurotransmission as assessed by using whole-cell patch-clamp recordings or indirectly by using biochemical assays of the sequelae of NMDA receptor-mediated calcium flux. The inhibitory effects of the enzyme are fully reversed by exogenously applied D-serine, which by itself did not potentiate NMDA receptormediated synaptic responses. Thus, D-serine is an endogenous modulator of the glycine site of NMDA receptors and fully occupies this site at some functional synapses. D-Amino acids play prominent roles in bacteria but have not been thought to occur in substantial quantity or to have any important function in vertebrates. Recently, techniques to distinguish isomers of amino acids in routine assays have led to the identification in some mammalian tissues of substantial amounts of at least two D-amino acids, D-serine and D-aspartate (1). Although D-aspartate is present in selected neuronal populations in the brain, it is concentrated mainly in glands, especially the epinephrine-containing cells of the adrenal medulla, the posterior pituitary, and the pineal gland (2-4).In contrast, D-serine occurs primarily in the brain, with highest concentrations in regions enriched in N-methyl-D-aspartate (NMDA) receptors (5-7). In these areas immunohistochemical studies have localized D-serine to protoplasmic astrocytes, which ensheathe nerve terminals especially in areas of the brain enriched in NMDA receptors (7). Stimulation of the kainate subtype of glutamate receptor releases D-serine from protoplasmic astrocytes (7).Because exogenous D-serine potentiates NMDA receptormediated neurotransmission (8-11) and D-[ 3 H]serine selectively binds to the glycine site (6), D-serine has been proposed as an endogenous ligand for the strychnine-insensitive glycine site of the NMDA receptor (6). Activation of NMDA receptors requires the presence of a coagonist, initially thought to be glycine (8,(12)(13)(14), and a glycine-selective recognition domain has been localized on NMDA receptors (15-17). However, D-serine is at least as potent as glycine as a coagonist at this site (8,10,14). In addition, immunohistochemical studies have revealed an overlapping distribution of D-serine and NMDA receptor immunoreactivity in forebrain (7). In the developing cerebellum, D-serine is localized to Bergmann glia that regulate granule cell migration during development via NMDA receptors (7). In contrast, glycine immunoreactivity is localized differently from that of NMDA receptors except in the brainstem, where it closely parallels the distribution of NMDA receptors (7). Extracell...

R ecently, free D-serine and D-aspartate have been reported in mammals, especially in the nervous system (1, 2). Using highly selective antibodies we localized D-serine to protoplasmic astrocytes in the gray matter areas enriched in N-methyl-Daspartate (NMDA) receptors for the neurotransmitter glutamate (3, 4). We also showed that glutamate, acting through non-NMDA receptors, releases D-serine from astrocyte cultures (3). NMDA receptors require coactivation at a ''glycine site'' (5) at which D-serine is up to three times more potent than glycine (6-8), suggesting that D-serine is an endogenous ligand for this site, and is released by glutamate from astrocytic processes that ensheath the synapse. Extracellular levels of endogenous Dserine are comparable to glycine in prefrontal cortex, whereas in the striatum, extracellular D-serine Using partial amino acid sequence from our purified preparation of an enzyme from rat brain converting L-serine to D-serine (10), we now report cloning and expression of serine racemase and its localization to astroglia. Materials and MethodsCloning. Full-length serine racemase was cloned by reverse transcription-PCR from mouse brain mRNA using primers based on mouse expressed sequence tags (ESTs) 615391 and 464586 (GenBank accession numbers A A170919 and AA032965, respectively), which corresponded to the 5Ј and 3Ј ends of the gene: forward primer, 5Ј-ATG TGT GCT CAG TAC TGC ATC TCC-3Ј; reverse primer, 5Ј-TTA AAC AGA AAC CGT CTG GTA AGG-3Ј. Several other mouse ESTs also covered parts of the sequence of serine racemase (GenBank accession numbers AI322578, AI173393, AA833469, and AA197364). The ORF and stop codon were confirmed by an independent 3Ј rapid amplification of cDNA ends reaction using a primer against 5Ј untranslated region (5Ј-AAA CAC AGG AGC TGT CAG C-3Ј). The mouse serine racemase sequence was deposited in GenBank (accession number AF148321).Cell Culture and Transfection. HEK293 cells were cultured in DMEM͞penicillin-streptomycin͞10% FBS media. Cells were transfected with serine racemase constructs subcloned in pRK5-KS vector with a cytomegalovirus promoter (provided by A. Lanahan and P. Worley, Johns Hopkins University) by using the calcium chloride method. Serine racemase mutant K56G was constructed by PCR. Equal expression of wild-type and mutant enzyme was confirmed by Western blot analysis of transfected cells. D-Serine Synthesis.In studies of D-serine synthesis, cells were cultured in media supplemented with increasing concentrations of L-serine. Unsupplemented media contained 0.4 mM L-serine. L-serine used in the experiments was rendered free of contaminating D-serine as described (10). D-serine in cells was measured in 25-mm culture dishes 36 hr after transfection. The cells were washed twice with cold PBS, followed by addition of 5% trichloroacetic acid (TCA) to extract free amino acids. After removing TCA with diethyl-ether, D-serine content was analyzed by both HPLC and a luminescence method as described (10). For measurement of D-serine in culture media, an a...

Significance Communication between nerve cells occurs at specialized cellular structures known as synapses. Loss of synaptic function is associated with cognitive decline in Alzheimer’s disease (AD). However, the mechanism of synaptic damage remains incompletely understood. Here we describe a pathway for synaptic damage whereby amyloid-β 1–42 peptide (Aβ 1–42 ) releases, via stimulation of α7 nicotinic receptors, excessive amounts of glutamate from astrocytes, in turn activating extrasynaptic NMDA-type glutamate receptors (eNMDARs) to mediate synaptic damage. The Food and Drug Administration-approved drug memantine offers some beneficial effect, but the improved eNMDAR antagonist NitroMemantine completely ameliorates Aβ-induced synaptic loss, providing hope for disease-modifying intervention in AD.

Haem oxygenase-1 (HO1) is a heat-shock protein that is induced by stressful stimuli. Here we demonstrate a cytoprotective role for HO1: cell death produced by serum deprivation, staurosporine or etoposide is markedly accentuated in cells from mice with a targeted deletion of the HO1 gene, and greatly reduced in cells that overexpress HO1. Iron efflux from cells is augmented by HO1 transfection and reduced in HO1-deficient fibroblasts. Iron accumulation in HO1-deficient cells explains their death: iron chelators protect HO1-deficient fibroblasts from cell death. Thus, cytoprotection by HO1 is attributable to its augmentation of iron efflux, reflecting a role for HO1 in modulating intracellular iron levels and regulating cell viability.

High levels of D-serine occur in mammalian brain, where it appears to be an endogenous ligand of the glycine site of N-methyl-D-aspartate receptors. In glial cultures of rat cerebral cortex, D-serine is enriched in type II astrocytes and is released upon stimulation with agonists of non-N-methyl-D-aspartate glutamate receptors. The high levels of D-serine in discrete areas of rat brain imply the existence of a biosynthetic pathway. We have purified from rat brain a soluble enzyme that catalyzes the direct racemization of L-serine to D-serine. Purified serine racemase has a molecular mass of 37 kDa and requires pyridoxal 5-phosphate for its activity. The enzyme is highly selective toward L-serine, failing to racemize any other amino acid tested. Properties such as pH optimum, K m values, and the requirement for pyridoxal phosphate resemble those of bacterial racemases, suggesting that the biosynthetic pathway for D-amino acids is conserved from bacteria to mammalian brain.D-Amino acids are prominent in bacteria whereas in animal tissues L-amino acids occur exclusively, though there have been occasional reports of D-amino acids, usually in invertebrates (1, 2). Recently, D-serine (3-6) and D-aspartate (7,8) were reported in mammalian tissues, especially in the nervous system. Using highly selective antibodies, we localized D-aspartate to neuroendocrine tissues (9), whereas the immunohistochemical localizations of D-serine closely resemble N-methyl-D-aspartate (NMDA) receptors for the neurotransmitter glutamate, as the distribution of D-serine measured chemically (10, 11). Glutamate cannot activate the NMDA receptor in the absence of glycine, indicating a ''glycine site'' for the receptor (12, 13). D-Serine is up to three times more potent than glycine at this site (14), suggesting that D-serine is the endogenous ligand for this site. D-Serine is localized exclusively to type II astrocytes, a form of glia concentrated in gray matter in the same areas of the brain as NMDA receptors (10). Stimulation of the kainate subtype of glutamate receptors releases D-serine from type II astrocytes, implying that synaptic release of glutamate triggers release of D-serine from the astrocytes to activate NMDA receptors physiologically (10). Although in most parts of the brain the distribution of D-serine resembles NMDA receptors far better than glycine, in some areas glycine and NMDA receptors are colocalized, suggesting that D-serine is the predominant ligand for the receptor in most brain areas but that glycine serves this purpose in some sites (11).Understanding the neurobiology of D-serine requires delineation of its biosynthesis. D-Serine might be formed by direct racemization from L-serine. Dunlop and Neidle (15) reported the transformation of radiolabeled L-serine to D-serine in intact rats, but this might have involved multiple steps rather than any direct enzymatic racemization. In the present study we describe an enzyme activity that directly racemizes L-serine to D-serine and that is localized to the brain. We ...

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...

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