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Neurobiology of DAmino Acids

E. Dumin . H. Wolosker

1 2 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 Neurobiology of DSerine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 A Physiological Regulator of NMDA Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 209 Serine Racemase: Biosynthesis of DSerine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 211 Regulation of DSerine Production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 DSerine Metabolism and Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 212 DSerine Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214 New Roles for DSerine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 DSerine and NMDA Receptor Dysfunction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215 Neuromodulator or Neurotransmitter? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
DSerine:

3 Neurobiology of DAspartate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 3.1 DAspartate in the Nervous and Endocrine System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 217 3.2 Role of Endogenous DAspartate: Still Mysterious After Two Decades . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 3.3 DAspartate Transport and Release . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 3.4 Biosynthesis of DAspartate . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218 3.5 DAspartate Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 3.6 DAspartate as Precursor for Endogenous NMDA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219 4 Conclusions and perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 219

Springer-Verlag Berlin Heidelberg 2007

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Neurobiology of Damino acids

Abstract: DAmino acids are generally perceived as unnatural isomers and are thought not to play a role in mammals. In the recent years, data obtained from several laboratories established the occurrence of large amounts of Dserine and Daspartate in the mammalian brain, where they seem to play unique roles. Endogenous Dserine is a highafnity ligand of the glycine site of NMDA receptors and is required for NMDA receptor activation. Synthesis of endogenous Dserine is catalyzed by serine racemase enzyme, which directly converts L into Dserine in the brain. DAspartate is enriched in neuroendocrine tissues and has been implicated in the regulation of hormone synthesis and release. DSerine and Daspartate are substrates for plasma membrane amino acid transporters and metabolized by specic peroxisomal oxidases. We now review several aspects of the neurobiology of these Damino acids, including their actions, target receptors, metabolic pathways, and roles in pathological conditions related to NMDA receptor dysfunction. List of Abbreviations: AMPA, a-Amino-3-Hydroxy-5-Methylisoxasole-4-Propionic Acid; NMDA, N-Methyl D-Aspartate; PLP, Pyridoxal 50 -Phosphate

Introduction

The origin of life on Earth is intimately related to the selection of stereochemical favorable compounds. Except for glycine, all common amino acids exhibit a chiral center resulting in the occurrence of two different enantiomers (L and D). Although the chemical properties of L and Damino acids are identical, Lamino acids were selected as the protein-building blocks as proteins are synthesized exclusively from L and not Damino acids. The sporadic presence of Damino acid residues in proteins is due to posttranslational modications in aged proteins rather than direct incorporation during translation (Fujii et al., 1999; Young et al., 2005). The presence of Damino acids in bacteria has been recognized a long time ago, where they constitute the bacterial cell wall, making it more resistant to proteases (Izaki et al., 1968). Later studies identied free Damino acids in the cytosol of invertebrate cells, but their role remained elusive (Srinivasan et al., 1962; Corrigan, 1969; DAniello and Giuditta, 1978). Till recently, Damino acids were thought not to exist in substantial quantities in higher organisms, and little or no attention has been directed toward their study. In recent years, remarkable quantities of Damino acids were discovered in the mammalian brain, especially Dserine and Daspartate (Dunlop et al., 1986; Fisher et al., 1991; Hashimoto et al., 1993a). They are not incorporated into proteins or peptides, constituting a free amino acid pool in the brain. In the last few years, there has been considerable progress in the understanding of the role of Dserine and Daspartate in the brain. There is experimental evidence that these enantiomers play important neurobiological roles, with implications for the pathophysiology of human diseases. The biosynthetic enzyme for Dserine in the brain has been discovered (Wolosker et al., 1999a, b; De Miranda et al., 2000), and Dserine seems to be a major modulator of NMDA receptor transmission (Mothet et al., 2000). DAspartate role is less clear and it has been linked to the regulation of hormone release (DAniello et al., 1996, 2000b; Takigawa et al., 1998; Wang et al., 2000). In the following sessions, we discuss several aspects of Damino acid distribution and role in the nervous system, as well as their recently discovered biosynthetic and metabolic pathways. Although several Damino acids exist at trace levels in the brain, we now review only Dserine and Daspartate since these are the only Damino acids present at signicant levels (Wolosker et al., 2002). The study of Damino acids in the brain can now be considered an exciting new eld in molecular neuroscience.

Neurobiology of DSerine

has been shown to occur at high levels in the brain, where it is an endogenous coagonist of NMDA receptors. > Table 9-1 summarizes the properties and disposition of Dserine, which suggest a major role for this Damino acid in the nervous system.

DSerine

Neurobiology of Damino acids . Table 9-1 Disposition and properties of Dserine

DSerine

209

Occurrence Cellular distribution Action Receptor Biosynthesis Metabolism Transport Release

Enriched in the mammalian brain Astroglia and some neurons Endogenous coagonist of NMDA receptor Coagonist site of NMDA receptors Serine racemase Peroxisomal Damino acid oxidase, a,belimination catalyzed by serine racemase Neutral amino acid transporters Elicited by glutamate through reversal of ASCTlike transporter and vesicular release from cultured astrocytes Adjuvant for neuroleptic treatment in schizophrenia PLP inhibitors, Lserine Osulfate, cysteine, phenazines

Potential therapeutic use Biosynthesis inhibitors

References Hashimoto et al. (1993a, 1992a) Schell et al. (1995, 1997a), Kartvelishvily (2006) Mothet et al. (2000) Kleckner and Dingledine (1988) Wolosker et al. (1999a, b), De Miranda et al. (2000) Nagata (1992), Foltyn et al. (2005) Hayashi et al. (1997), Ribeiro et al. (2002) Schell et al. (1995), Ribeiro et al. (2002), Mothet et al. (2005) Tsai et al. (1998), HerescoLevy et al. (2005) Wolosker et al. (1999b), Panizzutti et al. (2001), Kim et al. (2005)

2.1 DSerine: A Physiological Regulator of NMDA Receptors

NMDA receptors are one of the most important neurotransmitter receptors in the central nervous system. Broadly distributed throughout the brain, they play a major role in excitatory neurotransmission. NMDA receptor mediates Ca2 inux that is required for neuronal plasticity, development, leaning, and memory (Kemp and McKernan, 2002). Knockout of NMDA receptor subunit NR1 leads to embryonic lethality, demonstrating its prominent role in CNS (Forrest et al., 1994). A unique feature of NMDA receptors relates to the requirement of more than one agonist for the channel to operate. In addition to glutamate, binding of glycine to the receptor is essential for NMDA receptor function (Johnson and Ascher, 1987; McBain et al., 1989). Glycine binding to NMDA receptor is insensitive to strychnine, and this coagonist site is generally referred to as the glycine site. The coagonist site is located at the NR1 subunit, while the glutamate/NMDA site is found at the NR2 subunit. The afnity of NMDA receptors to glycine is in the low micromolar range (Matsui et al., 1995; Danysz and Parsons, 1998). Due to the high values of extracellular glycine concentration, it has been assumed that the glycine site of NMDA receptors will always be saturated and that this site would not play a dynamic role in NMDA neurotransmission (Danysz and Parsons, 1998). While there are several experimental examples in which the glycine site is saturated in vitro (Kemp et al., 1988; Fletcher et al., 1989; Mothet et al., 2000), this seems to be an exception to the rule. A large number of studies clearly show that the glycine site is not saturated under most experimental conditions both in vitro and in vivo (Wood et al., 1989; Danysz and Parsons, 1998). This implies that the glycine site of NMDA receptors may play an important role in regulating glutamatergic neurotransmission. Another aspect of the regulatory role of the glycine site relates to the identity of the endogenous coagonist. Early studies have shown that, similar to glycine, Dserine binds to the coagonist site of NMDA receptors with high afnity (Kleckner and Dingledine, 1988; McBain et al., 1989). Since this Damino acid was considered to be unnatural, Dserine was not thought to physiologically regulate NMDA receptors. This notion was overturned when Hashimoto et al. (1992a, 1993a) discovered large quantities of Dserine in

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the mammalian brain. Levels of Dserine in the brain are about onethird those of Lserine, while peripheral tissues contain only trace amounts of Dserine. Moreover, brain levels of Dserine are comparable or higher than many common Lamino acids such as asparagine, isoleucine, valine, and tryptophan (Hashimoto et al., 1992b). This led Hashimoto et al. (1992a) to suggest that Dserine could be an endogenous coagonist of NMDA receptors. The possibility that Dserine was an endogenous coagonist of NMDA receptors was soon strengthened by detailed immunohistochemical studies carried out by Snyder group (Schell et al., 1995, 1997a). Using a selective antibody against Dserine, Schell et al. (1995) found that Dserine is enriched in areas containing highest levels of NMDA receptors, including the rostral cerebral cortex, hippocampus, and striatum. DSerine densities are much less in the caudal part of the brain, including the adult cerebellum. By contrast, glycine immunoreactivity is higher in caudal areas of the brain, where densities of NMDA receptors are lower (Schell et al., 1997a). The inverse localizations of Dserine and glycine led Schell and coworkers to propose that endogenous Dserine was closer to NMDA receptors than glycine. Moreover, the authors found that Dserine densities were localized in astrocytes in the gray matter, raising the possibility that Dserine would be released from astrocytes ensheathing the synapse to activate neuronal NMDA receptors (Schell et al., 1995; Snyder and Ferris, 2000). Additional data support the notion that Dserine is a relevant endogenous coagonist of NMDA receptors. Microdialysis experiments demonstrated that extracellular Dserine concentration is similar or higher than glycine in some brain areas such as cerebral cortex and striatum (Hashimoto et al., 1995, 2000; Ciriacks and Bowser, 2004). The threedimensional structure of Dserine is similar to that of glycine, and its afnity for NMDA receptors is comparable or even higher than glycine (Matsui et al., 1995). A structural explanation for this nding comes from the analysis of Xray structures of the binding core of NR1 subunit of NMDA receptors (Furukawa and Gouaux, 2003). The crystal structures revealed that agonist binding is critically dependent on a series of hydrogen bonds to sidechain and mainchain atoms, as well as to water molecules. Furukawa and Gouaux (2003) proposed that Dserine binds more tightly to the receptor in comparison with glycine because it makes three additional hydrogen bonds and displaces a water molecule. Interestingly, Lserine binds 300fold less tightly than Dserine on NMDA receptors. Modeling of Lserine binding using Dserine as a guide showed that the hydroxyl group of Lserine unfavorably interacts in the binding pocket, providing astonishing Disomer specicity (Furukawa and Gouaux, 2003). More direct evidence that NMDA receptor is the target for endogenous Dserine came from experiments utilizing Damino acid oxidase treatment to selectively deplete endogenous Dserine in hippocampal cell cultures (Mothet et al., 2000). DAmino acid oxidase specically degrades D but not Lamino acids or glycine (Mattevi et al., 1996). Thus, the application of puried Damino acid oxidase preparation led to depletion of endogenous Dserine without affecting glycine levels in hippocampal primary cultures. Depletion of Dserine led to a 60%70% decrease in spontaneous activity attributed to postsynaptic NMDA receptor, while AMPA responses were unaffected (Mothet et al., 2000). Although the demonstration that Dserine is an endogenous coagonist of NMDA receptors in cell cultures was clear, its role in a more physiological preparation was uncertain. DAmino acid oxidase treatment was much less effective in hippocampal slices, in which it only depleted about 19% of total Dserine (Mothet et al., 2000), precluding any conclusion about the role of Dserine in this preparation. Subsequent studies conrmed that addition of Damino acid oxidase led to a decrease in NMDA receptor responses in the retina and in longterm potentiation of synaptic activity in the hippocampus (Stevens et al., 2003; Yang et al., 2003, 2005; Miller, 2004). Despite the reported ineffectiveness of Damino acid oxidase in hippocampal slices, these studies did not check if Dserine levels were actually depleted after Damino acid oxidase treatment. One concern with experiments employing Damino acid oxidase is that it displays very low afnity for Dserine (about 50 mM at pH 7.4) (DAniello et al., 1993b). In addition, commercial preparations of Damino acid oxidase may contain many impurities, including Daspartate oxidase, which quickly degrades NMDA (Shleper et al., 2005). Thus, unless levels of Dserine are not carefully monitored, it is not possible to evaluate the specicity of the treatment with Damino acid oxidase. Recently, a new developed technique overcame many caveats encountered with the use of Damino acid oxidase as a tool to remove Dserine. The bacterial Dserine deaminase enzyme degrades Dserine to pyruvate

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and ammonia (Marceau et al., 1988), being three orders of magnitude more efcient than Damino acid oxidase as judged by its kinetic parameters. By using this enzyme, it was possible to completely remove endogenous Dserine from hippocampal organotypic slices preparation (Shleper et al., 2005). Depletion of endogenous Dserine in organotypic slices virtually abolished NMDA receptorelicited neurotoxicity. By contrast, depletion of Dserine did not affect kainate neurotoxicity. This indicates that endogenous Dserine is the dominant and necessary coagonist for NMDA receptor neurotoxicity. Endogenous Dserine may also regulate important developmental processes. During cerebellar development, granule cells migrate from the external to the internal granule cell layer (Komuro and Rakic, 1993). Bergman glia, which contains high levels of endogenous Dserine, serves as scaffold for granule cell migration. Blockage of NMDA receptor at granule cells decreases the rate of migration (Komuro and Rakic, 1993). Recent observation suggests that endogenous Dserine presumably released by Bergman glia mediates the NMDA receptordependent neuronal migration in the cerebellum (Kim et al., 2005). Since migrating granule cells do not make conventional synaptic connections, the modulatory action of glialreleased Dserine reects a novel mechanism for neuromodulation (Kim et al., 2005).

2.2 Serine Racemase: Biosynthesis of DSerine

Because amino acid racemases were thought not to exist in mammals, the origin of brain Dserine has been a mystery. The most compelling evidence for an important role for Dserine came from the identication of a specic biosynthetic enzyme. The initial studies of the origin of Dserine were contradictory. Patients lacking the ability to degrade glycine by mutations in the glycine cleavage system (GCS) had decreased levels of brain Dserine, suggesting the possible involvement of GCS in Dserine synthesis (Iwama et al., 1997). On the other hand, administration of Lserine to rats increases brain Dserine, implicating Lserine as a precursor for Dserine synthesis (Dunlop and Neidle, 1997; Takahashi et al., 1997). The biosynthetic pathway from Lserine was established by the purication and cloning of serine racemase, an enzyme that converts L to Dserine in rat (Wolosker et al., 1999a, b; Panizzutti et al., 2001) and human brain (De Miranda et al., 2000). Serine racemase comprises 339 amino acids with a predicted molecular weight of 36.3 kDa (Wolosker et al., 1999b). The enzyme utilizes pyridoxal 50 phosphate (PLP) as cofactor and displays highsequence homology with the foldtype II group of PLP enzymes such as serine/threonine dehydratase (De Miranda et al., 2000). Serine racemase does not bear signicant homology to bacterial racemases. The enzyme occurs in postnatal glia but was also present at lower levels in prenatal neurons (Wolosker et al., 1999b). Transfection of serine racemase gene conferred to mammalian-cultured cells the ability to synthesize large amounts of Dserine. The distribution of serine racemase is closely similar to that of endogenous Dserine, with highest concentrations in the forebrain. The racemization of L into Dserine catalyzed by serine racemase was initially thought to be solely dependent on PLP (Wolosker et al., 1999b). Accordingly, PLP forms a Schiff s base with the lysine56 (Lys56) and mutations of this amino acid abolished serine racemase activity (Wolosker et al., 1999a). Subsequent studies showed that magnesium and ATP are physiological ligands of the enzyme and stimulate the racemization of Lserine several fold (De Miranda et al., 2002; Foltyn et al., 2005). Calcium also binds and activates serine racemase (Cook et al., 2002), but the afnity for calcium is much lower than for magnesium, indicating that magnesium rather than calcium is the physiological cofactor (De Miranda et al., 2002). Magnesium stimulates serine racemase by two distinct mechanisms: the rst is related to a direct binding of magnesium to the enzyme and the second mechanism involves an increase in the afnity to ATP due to the formation of a MgATP complex (De Miranda et al., 2002). The cofactors magnesium and ATP also disclosed new reactions catalyzed by serine racemase as they strongly stimulate the a,belimination of water from Lserine to form pyruvate and ammonia (De Miranda et al., 2002; Neidle and Dunlop, 2002). For each molecule of Dserine synthesized, about three to four molecules of pyruvate are formed in the presence of magnesium and ATP (De Miranda et al., 2002; Foltyn et al., 2005). It has been proposed that generation of pyruvate by the elimination activity of serine racemase within astrocytes may play a metabolic role by providing extraenergy in situations of increased energy demands

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(De Miranda et al., 2002). This is the rst indication that serine racemase is linked to energy metabolism. However, further studies on the role of serine racemasederived pyruvate will be required to clarify its role in cellular metabolism. LThreonine is also a substrate for serine racemase both in vitro and in vivo. Elimination of water from Lthreonine is strictly dependent on ATP, generating 2oxobutyrate and ammonia (Foltyn et al., 2005). Different from Lserine, threonine is not epimerized by serine racemase. The physiological role of Lthreonine utilization by serine racemase is not clear. The a,belimination catalyzed by serine racemase can be explained by the known reactions catalyzed by PLP (Morino and Snell, 1967; Schnackerz et al., 1979; Faraci and Walsh, 1988). > Figure 9-1 describes the chemical mechanism for racemization and a,belimination of Lserine (Foltyn et al., 2005). PLP is bound to the enzyme through an internal aldimine with Lys56. Reaction with the substrate Lserine generates an external aldimine intermediate. Subsequent aproton abstraction forms a resonancestabilized carbanion, which has a planar conguration. Reprotonation of this intermediate on the opposite face of the planar carbanion generates the Dserine external aldimine intermediate. DSerine is released via transimination with Lys56, regenerating the free enzyme. The resonancestabilized carbanion is also an intermediate for the a,belimination reaction. Elimination of the bhydroxyl group from the carbanionic intermediate leads to the formation of the aminoacrylatePLP intermediate. Subsequent transimination releases the initial aminoacrylate product and regenerates free enzyme. The aminoacrylate released undergoes rapid nonenzymatic hydrolysis to give pyruvate and ammonia (> Figure 9-1).

2.3 Regulation of DSerine Production

Modulation of serine racemase activity may be crucial for regulating brain Dserine levels and consequently NMDA receptor neurotransmission. Serine racemase protein does not contain any obvious regulatory region, though it contains potential sites for phosphorylation by protein kinase C and casein kinase. Recently, Snyder and coworkers showed that the glutamate receptor-interacting protein (GRIP1) binds to and activates mouse serine racemase (Kim et al., 2005). The interaction is mediated by the PDZ domain 6 of GRIP1 and the last four Cterminal amino acid of serine racemase. GRIP1 was found in astrocytes, and its overexpression strongly stimulates Dserine synthesis and enhanced granule cell migration in the cerebellum in vivo (Kim et al., 2005). Additionally, AMPA receptor stimulation increases Dserine synthesis and mediates granule cell migration in the developing cerebellum. After AMPA receptor activation, GRIP1 seems to dissociate from the receptor and binds to mouse serine racemase, activating the enzyme. The molecular mechanism by which GRIP1 activates mouse serine racemase is not clear, and it may result either from a direct effect of GRIP1 on the enzyme or due to recruitment of additional interacting proteins. Nevertheless, this study provides the rst mechanism by which a transmitter signal may alter Dserine levels, with implications for important developmental processes such as neuronal migration (Kim et al., 2005). Curiously, the cloned rat serine racemase gene lacks the Cterminal PDZ-binding motif found in the mouse enzyme (Konno, 2003). This indicates that regulation of serine racemase enzyme may signicantly differ among rodent species.

2.4 DSerine Metabolism and Transport

Brain Dserine exhibits a halflife of about 16 h (Dunlop and Neidle, 1997). The only enzyme known to degrade Dserine in mammals is Damino acid oxidase, which occurs in the cerebellum and brainstem. Metabolism of Dserine by Damino acid oxidase generates hydroxypyruvate, H2O2, and NH3. In the brain, the enzyme is mainly expressed in astrocytes of the cerebellum and brain stem (Horiike et al., 1994; Urai et al., 2002). Mice carrying a mutation in Damino acid oxidase gene exhibit augmented levels of Dserine in the cerebellum, brain stem, and spinal cord, demonstrating that Damino acid oxidase physiologically metabolizes Dserine (Nagata, 1992; Hashimoto et al., 1993a). These mice display higher sensitivity to

. Figure 9-1 Reaction mechanism of Lserine racemization and a,belimination. For details, see text and Foltyn et al. (2005). Reproduced with permission

Neurobiology of Damino acids

9
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pain caused by exacerbation of NMDA receptor activity in the spinal cord dorsal horn neurons (Wake et al., 2001). DAmino acid oxidase, however, occurs only at very low levels in the forebrain areas, such as the cerebral cortex and hippocampus, where high levels of Dserine are quite constant from 3 to 86 weeks old rats. Moreover, mutant mice possessing inactive Damino acid oxidase enzyme exhibit large increases in Dserine in the cerebellum and brain stem but do not display increased Dserine in forebrain areas (Hashimoto et al., 1993b). This strongly suggests that Dserine is not signicantly degraded by Damino acid oxidase enzyme in the forebrain. Recently, it has been shown that serine racemase catalyzes the degradation of cellular Dserine itself, through the a,belimination of water and subsequent production of pyruvate and ammonia. a,bElimination with Dserine is observed both in vitro and in cells (Foltyn et al., 2005). Thus, addition of Dserine to cells overexpressing serine racemase or astrocytes infected with lentivirus containing serine racemase leads to increased metabolism of Dserine (Foltyn et al., 2005). Several serine racemase mutants at a exible loop of serine racemase display impairment of a,belimination activity and increased racemization activity. This suggests a novel role for the a,belimination reaction, which is to limit the achievable Dserine concentration. a,bElimination also competes with the reverse serine racemase reaction (conversion of D into Lserine). No signicant reversal is observed with physiological levels of Dserine due to the transformation of Dserine into pyruvate (Foltyn et al., 2005). It has been proposed that elimination of water from Dserine may be important to limit Dserine levels in brain areas where Damino acid oxidase is poorly expressed (Foltyn et al., 2005). On its synthesis and release from the cells, Dserine stimulates NMDA receptors at the coagonist site. DSerine action could be terminated by Dserine reuptake into the cells. It is possible that serine racemase further degrades excess Dserine taken up into the cells through its a,belimination activity. This might constitute a mechanism for regulating intracellular Dserine concentration upon Dserine reuptake. One caveat of such model is that Dserine reuptake, rather than metabolism, is the key for termination of Dserine actions. Selective transporters for Dserine have not been identied yet, but at least two different neutral amino acid transport systems are candidates to mediate Dserine transport. The ASCT and asc1 types of neutral amino acid transporters can not only transport Dserine but also display high afnity for Lserine, Lalanine, Lcysteine, among others (Hayashi et al., 1997; Fukasawa et al., 2000; Nakauchi et al., 2000; Javitt et al., 2002; Ribeiro et al., 2002; Helboe et al., 2003). The afnity for Dserine has been reported to be 0.65 and 0.05 mM for ASCTlike and asc1 transporter, respectively (Nakauchi et al., 2000; Ribeiro et al., 2002). Despite its low afnity, the ASCTlike transporter present in astrocytes mediates Dserine release by heteroexchange mechanism (Ribeiro et al., 2002). While ASCTtype transporters are present in glia (Hayashi et al., 1997; Ribeiro et al., 2002), asc1 transporter is mainly neuronal (Helboe et al., 2003; Matsuo et al., 2004). Further studies on the regulation of Dserine transport and metabolism will shed light on the regulation of NMDA receptor coagonist levels in the brain.

2.5 DSerine Release

The mechanism of Dserine release has been studied using radioactive tracer or indirect determination of Dserine released in neural cultures. DSerine release from astrocyte cultures can be induced by glutamate, acting on AMPA/kainate receptors (Schell et al., 1995). Release of Dserine is also induced by heteroamino acid exchange catalyzed by ASCTlike transporters in astrocytes (Ribeiro et al., 2002). To date, no evidence exists on Dserine release catalyzed by reversal of the asc1 transporter. By using an indirect method with luminol chemiluminescence, vesicular release of Dserine from cultured astrocytes has been recently proposed (Mothet et al., 2005). The authors observed that AMPAelicited Dserine release was blocked by removal of calcium or treatment with botulinum toxin. This provides strong evidence that Dserine maybe released by a mechanism similar to that employed for conventional transmitters such as glutamate. One concern with the interpretation of luminol chemiluminescence is that the method is not linear and many drugs nonspecically affect luminescence. Moreover, one caveat of all the studies on Dserine release is the use of astrocyte cultures, which may not reect in vivo situation.

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A very promising approach to study the mechanism of Dserine release has been recently developed by Bowser group (Ciriacks and Bowser, 2004). Using capillary electrophoresis, the authors were able to measure endogenous Dserine release elicited by glutamate in brain slices. Further analysis of Dserine release in brain slices as well as direct demonstration of vesicular Dserine by immunoelectron microscopy will shed light on the mechanism of Dserine release.

2.6 New Roles for DSerine

New possible roles for serine racemase and Dserine come from recent localization studies. Serine racemase and Dserine occurs in microglia, and synthesis of Dserine can be induced by inammatory stimuli (Wu et al., 2004b). It has been proposed that Dserine released from microglia may mediate the neuronal cell death through overactivation of NMDA receptors that occurs after challenge with bamyloid (Wu and Barger, 2004; Wu et al., 2004b). Nevertheless, direct demonstration of Dserine and serine racemase in intact brain microglia is still missing. Serine racemase immunoreactivity was also demonstrated in Schwann cells of sciatic nerve preparation (Wu et al., 2004a, 2005). Since NMDA receptor does not occur along the sciatic nerve bers, the role of Dserine in the peripheral nervous system is different from that in the central nervous system. Serine racemase expression may be even wider than previously thought. Using a new antibody, we are able to detect expression of serine racemase also in neurons of the cerebral cortex (Kartvelishvily et al., 2006). This matches earlier observation that Dserine is detectable in some neurons of the cerebral cortex (Yasuda et al., 2001). This raises new questions on the role of Dserine and indicates a possible important role for the neuronal Dserine pool. For instance, Neuron-Derived Dserine can be released by KCl depolarization and ionotropic glutamate receptor activation. Moreover, Neuronal Dserine release mediates a signicant fraction of the NMDA receptor-elicited neurotoxicity in virtually pure neuronal cultures (Kartvelishvily et al., 2006).

2.7 DSerine and NMDA Receptor Dysfunction

As an endogenous coagonist of NMDA receptors, Dserine synthesized by serine racemase may play a role in several pathological conditions related to NMDA receptor dysfunction. NMDA receptor function is thought to be decreased in schizophrenia. The socalled NMDA receptor hypofunction hypothesis relies on clinical and animal observations. NMDA antagonists, such as phencyclidine, induce schizophrenic like symptoms in healthy volunteers and precipitate thought disorder and delusions in schizophrenia patients (Javitt, 2001; Coyle et al., 2003). In mice, Dserine antagonizes phencyclidine and MK801induced stereotyped behavior and ataxia (Contreras, 1990). Mice expressing only 5% of normal levels of NMDAR1 (NR1) subunit display behavioral abnormalities, including increased motor activity and stereotypy and decits in social and sexual interactions (Mohn et al., 1999). This condition is ameliorated by conventional antipsychotics. On the basis of the NMDA receptor hypofunction hypothesis, several clinical trials evaluated the effectiveness of NMDA coagonists in schizophrenia. Administration of Dserine greatly improved the negative, cognitive, and positive symptoms of the disease even when associated with conventional and new atypical antipsychotics (Tsai et al., 1998; HerescoLevy et al., 2005). Since the negative symptoms and cognitive impairment is normally resistant to conventional antipsychotic therapy, Dserine is a promising new adjuvant for schizophrenia treatment. Converging data indicate that endogenous Dserine metabolism may be altered in the disease as well. Using linkage analysis, Cohen et al. (2002) found association of the disease with single nucleotide polymorphisms (SNP) in the G72 gene. This gene interacts and activates Damino acid oxidase activity in vitro (Chumakov et al., 2002). Additionally, Cohen and coworkers found an association of schizophrenia with SNPs in Damino acid oxidase gene in a FrenchCanadian population. Association of schizophrenia with Damino acid oxidase polymorphisms was subsequently conrmed in German and Chinese

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population studies (Liu et al., 2004; Schumacher et al., 2004). In support of a role of Dserine in schizophrenia, a statistically signicant decrease in serum Dserine was described in patients in schizophrenic subjects (Hashimoto et al., 2003). Despite the signicant association of schizophrenia with SNPs in Damino acid oxidase gene, the genetic data should be interpreted with caution. Schizophrenia is a complex and multifactorial disease, in which several genes are likely to be involved. The contribution of a single gene may be very difcult to ascertain. Additional studies with larger populations will be required to establish a role of Damino acid oxidase in schizophrenia. Individual contribution of each gene may be small and difcult to quantify. Also, biochemical data on the activity of Damino acid oxidase in schizophrenic tissue will be crucial to determine its role in the disease. The overproduction of glutamate has been widely implicated in a large number of acute and chronic degenerative diseases. The harmful effects of excessive glutamate occur mainly through activation of the NMDA receptors and consequently by massive calcium inux into the cell (Danysz and Parsons, 1998). Since Dserine is an endogenous agonist of NMDA receptors, selective inhibitors of serine racemase will be valuable tools for investigating the regulation of NMDA transmission. Overstimulation of NMDA receptors is implicated in neural damage following stroke (Choi and Rothman, 1990). Elevated extracellular concentrations of Dserine are observed after transient cerebral ischemia in rats (Lo et al., 1998), and drugs that block the glycine site of NMDA receptors prevent stroke damage in animal models (Wenk et al., 1998). It has been proposed that inhibitors of serine racemase provide a new strategy to decrease NMDA receptor coactivation and may be useful in conditions, such as stroke and neurodegenerative diseases, where overstimulation of NMDA receptors play a pathological role (Panizzutti et al., 2001). On the other hand, a possible caveat in using serine racemase inhibitors to prevent stroke damage is the long halflife of Dserine in the brain. Conceivably, inhibitors of both racemization and a,belimination activities of serine racemase will block Dserine synthesis, but removal of preformed Dserine will be slow due to the absence of Damino acid oxidase activity in the forebrain. In this context, ligands that stimulate selectively Dserine a,b elimination while inhibiting racemization will be more effective in decreasing brain Dserine.

2.8 Neuromodulator or Neurotransmitter?

The coagonist site of NMDA receptors clearly modulates NMDA receptor responses. The afnity of NMDA receptor for glutamate increases by preexposure to coagonists Dserine or glycine (Benveniste et al., 1990; Lerma et al., 1990; Krasteniakov et al., 2005). Glycine and Dserine binding to NMDA receptors in the absence of glutamate also primes the receptors for internalization (Nong et al., 2003). Despite the ample evidence for the modulatory activity of the coagonist site, Dserine may be more than a neuromodulator. The operation of NMDA receptors requires the obligatory binding of the coagonist, otherwise the channel does not open (Johnson and Ascher, 1987). The term neuromodulator does not incorporate the obligatory presence of Dserine for NMDA receptor activity. Snyder and Ferris (2000) elegantly build the case for a transmitter role for Dserine by adopting a more liberal conceptualization of a neurotransmitter. The existence of metabolic pathways, a specic receptor, and mechanisms of transport and release point to a possible transmitter role for Dserine (> Table 9-1). Different from classical transmitters, Dserine has been thought to be exclusively made in glia. In support for a gliotransmitter role, it has been recently shown that Dserine release from cultured glial cells can occur through exocytosis (Mothet et al., 2005). Rapid release of Dserine was calcium dependent and induced by AMPA receptor stimulation. This provides a strong evidence that Dserine can function as a gliotransmitter (Mothet et al., 2005). The detection of Dserine and serine racemase in neurons of the cerebral cortex, however, indicates that Dserine also has a neuronal origin (Kartvelishvily et al., 2006). It has been suggested that neuron-derived Dserine stimulates NMDA receptors by an autocrine or Paracrine manner (Kartvelishvily et al., 2006). A common aspect of all transmitters is that their levels are dynamic and rapidly change at the synapse. To date, there is no experimental evidence on whether local Dserine levels are dynamic. Identication of

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specic signals that stimulate Dserine synthesis and/or release together with their effect on NMDA receptor neurotransmission will help to establish Dserine as a transmitter. Future studies that address this important issue will require the technical capability to directly measure tiny amounts of Dserine in a very fast timescale.

Neurobiology of DAspartate

DAspartate is the only other Damino acid reported to occur at signicant levels in the central nervous system. Lajtha and associates rst detected free Daspartate in mammalian brain and other tissues (Dunlop et al., 1986). Though aged proteins may exhibit a limited posttranslational racemization of L into Daspartate, brain Daspartate is not directly incorporated into proteins (Dunlop et al., 1986). > Table 9-2 summarizes the current knowledge on Daspartate disposition and possible roles in neuroendocrine tissues.

. Table 9-2 Disposition and properties of Daspartate

DAspartate

Occurrence Action Receptor Biosynthesis Metabolism Transport Release Biosynthesis inhibitors

Neuroendocrine tissues and developing neurons Not known, endocrine modulator? Unclear. Glutamate site of NMDA receptors? From Laspartate in cultured cells. Biosynthetic enzyme not known Peroxisomal Daspartate oxidase Highafnity excitatory amino acid transporters Elicited by acethylcholine, nicotine and KCl depolarization from adrenal slices PLP inhibitor

References Dunlop et al. (1986), Hashimoto et al. (1993a), Schell et al. (1997b), Wolosker et al. (2000) DAniello et al. (1996), DAniello et al. (2000b) Olverman et al. (1988) Long et al. (1998), Wolosker et al. (2000) Dixon and Kenworthy (1967) Karlsen and Fonnum (1978), Taxt and StormMathisen (1984) Wolosker et al. (2000) Wolosker et al. (2000)

3.1 DAspartate in the Nervous and Endocrine System

occurs early during brain development, its levels dramatically fall after birth (Dunlop et al., 1986; Hashimoto et al., 1993a). Immunohistochemistry with a selective antibody against Daspartate revealed that it occurs exclusively in neurons with no evidence of staining in glia (Schell et al., 1997b). DAspartate localizations are associated to regions that play a major role in regulating development. Particularly intense immunoreactivity occurs in the cortical plate and the subventricular zone at P0 (Wolosker et al., 2000). The hippocampus displays pronounced staining in pyramidal cells of CA1CA3 and the dentate gyrus. In the cerebellum, the most intense immunoreactivity occurs in the external granular layer associated with granule cells that have not yet undergone migration to the adult granule cell layer. Very high levels of Daspartate are also observed in embryonic primary neuronal cultures, where it accounts for almost half of total aspartate levels (Wolosker et al., 2000). In adult animals, little Daspartate is left in forebrain areas (Wolosker et al., 2000). Highest concentrations of Daspartate persist in the pineal, pituitary, adrenal glands, and testis (Dunlop et al., 1986; Lee et al., 1997, 1999; Imai et al., 1995; DAniello et al., 2000a). In the adrenal gland, Daspartate is conned to the medulla where it is concentrated specically in epinephrine cells (Schell et al., 1997b).
DAspartate

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3.2 Role of Endogenous DAspartate: Still Mysterious After Two Decades

Despite the abundance of Daspartate in neuroendocrine tissue, its role remains mysterious, as a specic receptor for endogenous Daspartate has not been identied yet. NMDA receptors bind Daspartate, but it is not clear whether endogenous Daspartate will activate NMDA receptors in vivo, as the afnity for Daspartate is several fold lower than for glutamate (Olverman et al., 1988). Recently, it has been shown that AMPA receptors are blocked by Daspartate, but not by the L enantiomer (Gong et al., 2005). DAspartate competitively blocks Lglutamate and kainate effects in rat hippocampal neurons and AMPA receptors expressed in Xenopus oocytes, but the afnity of AMPA receptors for Daspartate was much lower than for glutamate (Gong et al., 2005). Several studies suggested possible roles for Daspartate by employing exogenous administration of Daspartate. DAspartate greatly stimulates testosterone synthesis in rat testes and in isolated Leydig cells (DAniello et al., 1996, 1998, 2000b; Di Fiore et al., 1998; Sakai et al., 1998) by apparently increasing the messenger RNA levels of a steroidogenic acute regulatory protein (Nagata et al., 1999a, b). DAspartate administration to rats also enhances oxytocin (Wang et al., 2000), prolactin (DAniello et al., 2000b), and luteinizing hormone release (DAniello et al., 2000a). DAspartate taken up by pinealocytes depresses melatonin synthesis (Takigawa et al., 1998). Taken together, these studies suggest that Daspartate is an important endocrine modulator, but they do not provide any evidence that the endogenous Daspartate actually affects hormone synthesis and release. Further studies are needed to address the role of endogenous Daspartate. This will require the identication of its molecular targets and biosynthetic pathway. Techniques to deplete Daspartate from the tissues, similar to those previously employed for Dserine, will be valuable to access the role of endogenous Daspartate.

3.3 DAspartate Transport and Release

is taken by excitatory amino acid transporters, and its tritiated form has been extensively used in transport studies (Karlsen and Fonnum, 1978; Taxt and StormMathisen, 1984; Gundersen et al., 1995). Immunohistochemical studies showed that Daspartate is not taken up in synaptic vesicles (Gundersen et al., 1995). This seems to be due to the very high specicity of the vesicular glutamate transporters (Wolosker et al., 1996). DAspartate can also be mobilized from endogenous stores in vivo, implying an important role. Epinephrine is released from the adrenal in response to activation of nicotinic cholinergic receptors following ring of the splanchnic innervation of the adrenal (Seidler and Slotkin, 1976; Nussdorfer, 1996). In vivo treatment of rats with high doses of nicotine depletes catecholamine stocks in adrenals (Seidler and Slotkin, 1976). Nicotine treatment is also very effective in releasing Daspartate, which are reduced by 70% in the adrenal medulla after chronic nicotine treatment, with no signicant change in Laspartate or Lglutamate concentrations (Wolosker et al., 2000). Mobilization of endogenous Daspartate is also observed in adrenal slices after depolarization with KCl or acetylcholine treatment (Wolosker et al., 2000).
DAspartate

3.4 Biosynthesis of DAspartate

Endogenous origin of Daspartate was rst demonstrated by a developmental increase in Daspartate concentration in chicken egg (Neidle and Dunlop, 1990). Since the egg is a closed system, any increase in Daspartate should be due to a biosynthetic process. Evidence that Daspartate is synthesized in mammalian tissue comes from an ingenious experiment carried out by DAniello et al. (1998). The author carefully measured the concentration of Daspartate in the testicular venous blood and compared it to the peripheral blood. Values for Daspartate were 20 higher in the testicular venous blood, indicating that testis synthesize and secrete Daspartate into the blood in vivo (DAniello et al., 1998). Further evidence supporting the presence of a specic biosynthetic route for Daspartate comes from experiments in cell cultures. Cultured pheochromocytoma cells (PC12) release Daspartate to the culture

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medium over time, suggesting a biosynthetic pathway in cancer cells (Long et al., 1998). In neuronal primary cultures, Daspartate synthesis seems to be mediated by Laspartate racemization. Biochemical evidence for racemization comes from the detection of Daspartate synthesis from L[3H]aspartate in primary neuronal cultures of rat cerebral cortex (Wolosker et al., 2000). Addition of aminooxyacetic acid, an inhibitor of PLPdependent enzymes, inhibited Daspartate synthesis (Wolosker et al., 2000). This indicates that, like Dserine, a specic racemase toward Laspartate might exist. Nevertheless, attempts to biochemically purify an aspartate racemase have failed. Identication of aspartate racemase will be crucial to understand the biological role of this Damino acid. Despite the general believe that most of Daspartate is endogenous, one cannot discard that a signicant fraction of Daspartate has an exogenous origin or is carried away from its synthesis site to target tissues that do not possess a biosynthetic enzyme. Of notice is the occurrence of Daspartate in tissues that are blood brain barrier decient such as pituitary and pineal glands. Blood levels of Daspartate may be derived from Daspartate absorbed from the gut that escaped liver metabolism or from Daspartate synthesized elsewhere in the body (e.g., testis). Conceivably, Daspartate could be carried out by the blood to the pituitary and pineal gland, which possess a highafnity transporter that takes up extracellular Daspartate (Takigawa et al., 1998). Further studies will be required to evaluate if tissues like the pituitary and pineal gland synthesize their own Daspartate.

3.5 DAspartate Metabolism

Degradation of Daspartate is mediated by Daspartate oxidase enzyme, which is able to oxidize Daspartate, Dglutamate, and Nmethyl Daspartate (Dixon and Kenworthy, 1967; DAniello et al., 1993a). Like Damino acid oxidase, Daspartate oxidase is a peroxisomal enzyme, with highest expression in the kidney and liver (DAniello et al., 1993b). However, Daspartate oxidase does not use Dserine as substrate. Highest levels of this enzyme occur in areas with lowest Daspartate levels, suggesting that Daspartate oxidase degrades endogenous Daspartate (Schell et al., 1997b).

3.6 DAspartate as Precursor for Endogenous NMDA

In invertebrates, occurrence of NMDA has been documented, but till recently NMDA was thought not to exist in mammals (Sato et al., 1987; Todoroki et al., 1999). Interestingly, DAniello et al. (2000a) have shown that Daspartate administration to rats elicits NMDA accumulation in the pituitary gland (DAniello et al., 2000a). The authors demonstrated the existence of endogenous NMDA in rats by an enzymatic method using Daspartate oxidase followed by HPLC analysis. Most importantly, the authors reconstituted the methylation of Daspartate into NMDA with rat brain homogenates utilizing sadenosylmethionine as a methyl group donor (DAniello et al., 2000a). If conrmed by additional analytical methods, the presence of endogenous NMDA in the brain will be of great importance. The enzyme responsible for its biosynthesis has not been identied yet. Identication of the biosynthetic route for NMDA will be important to denitively establish the presence of endogenous biosynthesis of NMDA in the mammalian brain.

Conclusions and perspectives

The eld of Damino acids research experienced signicant advances in the last few years. Several studies demonstrated an important physiological role of Dserine in excitatory neurotransmission. The physiological role of endogenous Daspartate and its derivative NMDA is still unclear, but major breakthrough is expected when their biosynthetic enzymes will be isolated. DSerine can now be considered a novel transmitterlike molecule in the brain, and the study of its neurobiology will shed light on the regulation of NMDA receptor neurotransmission. The isolation of serine racemase opened a new era in Damino acid research and will allow the study of the mechanisms regulating NMDA receptor coagonist levels in the

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brain. Although much remains to be understood, the demonstration of a physiological role for Dserine and the presence of a biosynthetic apparatus in mammals overturn classic concepts in biology, which usually consider Damino acids as unnatural products. In light of the NMDA receptor dysfunction in several human diseases, Dserine is likely may be relevant in several pathological states and lead to new therapies. Blockers of Dserine synthesis may be useful for conditions, such as stroke and neurodegenerative diseases, in which NMDA receptor stimulation plays a pathological role. In sum, Damino acid research now constitutes an exciting new eld in neurobiology and biochemistry, which is likely to attract increasing numbers of investigators.

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