l -Serine-O-phosphate in the central nervous system

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Research Report L-Serine-O-phosphate

in the central nervous system

Jordan E. Antflick a , Sandra Vetiska a , Joan S. Baizer b , Yi Yao a , Glen B. Baker c , David R. Hampson a,d,⁎ a

Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, Ontario, Canada M5S 3M2 Department of Physiology and Biophysics, The University at Buffalo, Buffalo, NY, USA c Neurochemical Research Unit and Bebensee Schizophrenia Research Unit, Department of Psychiatry, University of Alberta, Edmonton, Alberta, Canada d Department of Pharmacology, University of Toronto, Toronto, Ontario, Canada b

A R T I C LE I N FO

AB S T R A C T

Article history:

L-serine-O-phosphate (L-SOP)

Accepted 30 August 2009

pathway and is also an agonist at the Group III metabotropic glutamate receptors (mGluRs).

Available online 9 September 2009

L-SOP

is the immediate precursor to L-serine in the serine synthesis

is produced by the enzyme phosphoserine aminotransferase (PSAT) and metabolized

to L-serine by phosphoserine phosphatase (PSP). Using a novel analytical procedure, we Keywords:

show that L-SOP is present in rat whole brain, and that in transfected cells, it is substantially

Bergmann glia

more potent than L-glutamate at the mGluR4 receptor subtype. Immunocytochemical

Group III receptor

analyses showed that the distributions of PSAT and PSP in the cerebral cortex,

D-serine, L-serine

hippocampus, and cerebellum were similar in the rat and macaque monkey brain. In the

L-phosphoserine

rat hippocampus, cells within the subgranular zone were co-labeled with anti-PSP and anti-

Metabotropic glutamate receptor

PSA-NCAM, a marker for neurogenic cells. In the cerebellar cortex, Purkinje neurons

mGluR4

expressed relatively high levels of both enzymes while robust expression of PSAT was also observed in the Bergmann glia. L-SOP released from Purkinje neurons or Bergmann glia could activate mGluR4 present on parallel fiber terminals. The presence of L-SOP in brain, its high potency at mGluR4, together with the restricted distributions of the synthetic and metabolic enzymes, suggest that L-SOP might act activate Group III metabotropic glutamate receptors in the CNS. © 2009 Elsevier B.V. All rights reserved.

1.

Introduction

L-serine has been shown to be a requisite growth factor for neurons (Savoca et al., 1995; Furuya et al., 2000), while D-serine is a co-agonist at the N-methyl-D-aspartate subtype of glutamate-gated ion channels (Mothet et al., 2000; Shleper et al., 2005), and may also act as a ligand at the GluRδ2

glutamate-like receptor (Naur et al., 2007). In mammalian cells L-serine is synthesized via the “phosphorylated pathway” which encompasses three enzymatic steps. In the first step, 3phosphoglycerate derived from glycolysis is metabolized to phosphohydroxypyruvate by 3-phosphoglycerate dehydrogenase (de Koning et al., 2003). In the second step phosphohydroxypyruvate is converted into L-serine-O-phosphate (L-SOP),

⁎ Corresponding author. Leslie Dan Faculty of Pharmacy, University of Toronto, 144 College St., Toronto, Ontario, Canada M5S 3M2. Fax: +1 416 978 8511. E-mail address: [email protected] (D.R. Hampson). Abbreviations: CNS, central nervous system; GFAP, glial fibrillary acidic protein; HEK-293, human embryonic kidney 293 cells; l-AP4, l-2amino-4-phosphonobutyric acid; l-SOP, l-serine-O-phosphate; MAP2, microtubule-associated protein 2; mGluR, metabotropic glutamate receptor; PBS, phosphate-buffered saline; PSAT, phosphoserine aminotransferase; PSP, phosphoserine phosphatase 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.08.087

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also known as L-phosphoserine, by the vitamin B6-dependent enzyme phosphoserine aminotransferase (PSAT; Hester et al., 1999). L-SOP is then dephosphorylated by phosphoserine phosphatase (PSP; Collet et al., 1999) to produce L-serine which is subsequently metabolized to glycine by the enzyme serine hydroxymethyltransferase, or isomerized to D-serine by serine racemase. L-SOP, and its close synthetic congener 2-amino-4-phosphonobutyric acid (L-AP4), are selective agonists at the Group III metabotropic glutamate receptors that include mGluR4, mGluR6, mGluR7, and mGluR8 receptors (Nicoletti et al., 1986; Naples and Hampson, 2001; Brauner-Osborne et al., 2007; Hampson et al., 2008; Niswender et al., 2008). There is a paucity of information available about brain levels of L-SOP. Previous studies have reported values that differ considerably from one another, ranging from several hundred nanomolar to millimolar concentrations (McIlwain and Bachelard, 1971; Kataoka et al., 1991; reviewed in Klunk et al., 1991; Goodnough et al., 1995). The higher concentrations of L-SOP in some studies are roughly similar to that of free L-serine in the brain. Considering the impermeability of the blood–brain barrier to L-SOP, such a high concentration in brain would suggest that the enzymes in this pathway would be highly active and widely distributed within the CNS. The importance of the L-SOP synthetic and metabolic enzymes in CNS development is illustrated by reports of genetic mutations in 3-phosphoglycerate dehydrogenase, PSAT, and PSP in humans. Although rare, mutations in all three enzymes produce a common series of clinical outcomes that include abnormal brain development (e.g. microcephaly), seizures, and mental retardation (de Koning et al., 2003; Hart et al., 2007). In addition, the L-SOP metabolizing enzyme PSP is enriched in proliferating neural progenitor cells and embryonic and hematopoietic stem cells (Nakano et al., 2007). It was also demonstrated that L-SOP inhibited neural stem cell/progenitor proliferation, enhanced neurogenic fate commitment, and improved neuronal survival (Nakano et al., 2007; Saxe et al., 2007). Interestingly, some of the effects of LSOP (and exogenous L-AP4) on neural progenitor cells appear to be mediated by the Group III metabotropic glutamate receptor, mGluR4 (Iacovelli et al., 2006; Saxe et al., 2007; Nakamichi et al., 2008). In the present study we examined the levels of L-SOP in rat brain, its relative potency at mGluR4, and the distributions of the PSAT and PSP proteins in the rat and monkey CNS. Based on the findings presented here, together with those of other studies, we hypothesize that L-SOP may activate mGluR4, and possibly other Group III mGluRs in the brain.

2.

Table 1 – Concentrations of L-SOP, L-serine, and D-serine in adult rat whole brain and cerebellum.

Whole brain Cerebellum

L-SOP

L-Serine

D—Serine

0.99 ± 0.05 (5.37 ± 0.25) 1.21 ± 0.08 (6.51 ± 0.41)

78.36 ± 1.97 (745.58 ± 18.77) 68.92 ± 5.81 (655.75 ± 55.24)

25.10 ± 0.45 (238.85 ± 0.25) 4.83 ± 0.19 (45.95 ± 1.78)

Concentrations in μg/g of tissue and in nanomoles/g of tissue in parentheses. Each value represents mean ± S.E.M. of six samples.

The concentration of L-SOP was lower than D-serine and much lower than L-serine. The levels of L-SOP in cerebellum were significantly higher (p < 0.001, t-test) compared to whole brain, while the levels of D-serine in cerebellum were only about 20% of the levels in whole brain (Table 1). L-serine levels were roughly similar in whole brain and cerebellum. Although previous studies have reported potency values for L-SOP in various functional assays at Group III mGluRs including mGluR4 (Gomeza et al., 1996; Thomsen et al., 1997; Schoepp et al., 1999), we re-examined this by conducting a simultaneous side-by-side comparison of L-SOP, L-glutamate, and the prototypical synthetic agonist L-AP4 on rat mGluR4 expressed in HEK-293 cells. The EC50 values were 0.52 ± 0.08 μM, 0.46 ± 0.04 μM, and 15 ± 3 μM for L-SOP, L-AP4, and Lglutamate respectively (Fig. 1).

2.2. Characterization of the PSAT and PSP antibodies and the relative abundance of the two proteins in tissues To assess recognition of PSP by the rabbit PSP antibody, recombinant rat PSP protein was expressed in isopropylthiogalactoside-induced Escherichia coli and cell lysates were examined on western blots. The PSP antibody strongly labeled the 25-kDa PSP monomer in the induced samples, whereas no signal was observed in the uninduced samples, or when the same samples were probed with the preimmune antisera (Fig. 2A). C-myc-tagged mammalian cDNA expression constructs of PSAT and PSP were transfected into HEK-293 cells and analyzed by western blotting. The anti-PSAT

Results

2.1. Quantitation of L-SOP in rat brain and comparative potency at mGluR4 L-SOP

levels were assessed in adult rat whole brain (forebrain, cerebellum, and brainstem) and in cerebellum only. For comparison, L-serine and D-serine levels were also measured simultaneously in the same samples. L-SOP levels in rat whole brain were about 1 μg per gram of tissue (equivalent to 5.4 μM).

Fig. 1 – Dose–response analysis of L-SOP, L-AP4, and L-glutamate at mGluR4 receptors expressed in transiently transfected HEK-293 cells. The EC50 values were 0.52 ± 0.08 μM, 0.46 ± 0.04 μM, and 15 ± 3 μM for L-SOP, L-AP4, and L-glutamate respectively. Each point represents the average of three independent experiments.

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Fig. 2 – Characterization of the anti-PSAT and anti-PSP antibodies and tissue distributions in the rat. (A) Western blot of PSP expression in E. coli after induction with IPTG. The blot was probed with the anti-PSP antibody; a band of 25 kDa was observed in the induced but not in the uninduced E. coli samples. The same samples probed with the preimmune serum displayed no immunoreactivity. (B) HEK-293 cells were transfected with 2 μg of PSP or PSAT cDNA and probed with either PSAT (upper) or PSP (lower) antibodies revealing immunoreactivity for both the endogenous and tagged versions of PSAT and PSP. (C) PSP expression (25 kDa) in tissue samples prepared from postnatal day 2 and adult rats. (D) Comparison of PSAT expression (40 kDa) in tissue samples prepared from postnatal day 2 (PN2) rats and adult rats. In panels C and D, equal amounts of protein (8 μg) were loaded in each lane and verified by blotting with GAPDH.

antibody and the anti-PSP antibody labeled the respective untagged endogenous proteins, and slightly higher molecular weight proteins corresponding to the c-myc-tagged proteins (Fig. 2B). Enzyme expression in brain and peripheral tissues was first examined on western blots of postnatal day 2 and adult rat tissue samples probed with the anti-PSP (Fig. 2C) and antiPSAT (Fig. 2D) antibodies. PSP was observed as the 25-kDa enzyme monomer in all tissues. The highest levels of expression of PSP in postnatal day 2 and adult rat tissues were in liver, kidney, and spleen. The monomer of the dominant β-isoform of PSAT at 40 kDa was

observed in all organs examined in both the adult and postnatal day 2 rats. PSAT was expressed at widely varying expression levels in different tissues; the highest levels were seen in heart, spleen, and especially the liver and kidney. On western blots of samples of forebrain and cerebellum, expression of PSAT and PSP was observed in postnatal day 2 and adult rats (Figs. 2C, D). The expression of PSP was lower in the adult forebrain and cerebellum compared to postnatal day 2. In some tissues, the PSAT antibody labeled a doublet. Although two alternatively spliced forms of PSAT have been reported in human tissues, α and β, (Baek et al., 2003), the PSAT antibody used here recognizes only the dominant β

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Fig. 3 – Analysis of PSAT and PSP expression in the somatosensory region of the cerebral cortex of the adult rat. (A) PSAT expression was observed in cells in all layers of the somatosensory cortex including layer V illustrated here. (B) Omission of the primary antibodies showing absence of labeling in the presence of the combined anti-rabbit and anti-mouse secondary antibodies. (C) Anti-PSP immunostaining of pyramidal neurons in layers II/III and V of the somatosensory cortex. (D) Higher magnification image of anti-PSP staining in layers II/III overlaid with DAPI to visualize nuclei. Arrow indicates an example of cytosolic cell body staining. (E) Cortical section showing double labeling with anti-PSP and anti-GFAP and demonstrating the absence of co-localization. (F) Double labeling of layer V pyramidal neurons with anti-PSP and the neuronal dendritic marker MAP2 where co-expression in the same neuron is evident (example shown at arrow). Scale bars = 50 μm.

form. Therefore, the PSAT doublet observed might correspond to either post-translationally modified and unmodified forms of the enzyme, or an as yet uncharacterized alternative splice

form. Although the level of expression of PSAT and PSP appeared to be relatively low on western blots of adult rat brain, this may have been due to the dilution of the whole

Fig. 4 – Expression of PSP in the adult rat hippocampal formation. (A) PSP expression was predominantly observed in the subgranular zone of the dentate gyrus (arrow) and pyramidal cells (arrowhead) of the hippocampal formation. (B–D) Higher magnification immunofluorescence images of the dentate gyrus labeled with anti-PSP (B) and anti-GFAP (C) and the merged image of PSP and GFAP (D). Regions of limited co-localization of GFAP and PSP are seen in panel D (example indicated by the arrow). (E–G) Higher magnification images showing the subgranular zone (SGZ) of the dentate gyrus labeled with anti-PSP (panel E), anti-PSA-NCAM (panel F) and the merged image of PSP and PSA-NCAM (panel G). The arrow in panel G denotes an example of a cell where co-localization is evident. The granular cell layer is identified by the letter G. H, hilus; scale bars= 100 μm.

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forebrain and cerebellum electrophoresis samples because immunocytochemical analyses demonstrated prominent expression in select populations of neurons in the adult rat and monkey central nervous system (see below and Figs. 3, 4, 5, and 7).

2.3.

Immunocytochemical analysis

Immunocytochemical analyses were carried out on thin (14 μm) sections of the adult rat brain (Figs. 3–4), postnatal day 2 rat brain (Fig. 6), and on thicker sections (50 μm) of adult macaque brain (Fig. 7). We focused on the distributions of PSAT and PSP in three brain regions: the cerebral cortex, hippocampus, and cerebellum. In the adult rat cerebral cortex, immunostaining for both PSAT and PSP was detected throughout all cortical layers. Within the somatosensory region of the cerebral cortex, PSAT was detected at similar levels in the cell bodies of pyramidal neurons in every layer (Fig. 3A), whereas PSP expression was more prominent in pyramidal neurons of layers II/III and layer V (Fig. 3C). PSAT (Fig. 3A) and PSP (Fig. 3D) demonstrated cytosolic labeling in pyramidal cell bodies and an absence of labeling in nuclei as indicated by lack of overlap with the DAPI nuclear stain. Some cells were stained with DAPI but were unstained with the PSP antibody indicating that not all the cells in this region expressed PSP (Fig. 3D). The proximal dendrites of pyramidal

neurons were also labeled with the anti-PSP antibody in many cells. No labeling was observed in tissue sections incubated with only the anti-chicken or anti-rabbit secondary antibodies (Fig. 3B). Moreover, pre-adsorption of the anti-PSP antibody with a saturating amount of peptide abolished immunoreactivity (result not shown). In double labeling experiments, virtually no overlap of PSP with the glial cell marker, GFAP (Fig. 3E) was observed. In contrast, the dendrites of cells in which the cell bodies and proximal dendrites were labeled with antiPSP were immunostained with the neuronal dendritic marker MAP2 (Fig. 3F), indicating neuronal localization of PSP in the cerebral cortex. Other regions of the cerebral cortex displayed the same general pattern of immunostaining for both PSAT and PSP as shown in Fig. 3 for the somatosensory cortex (data not shown). In the rat hippocampal formation, immunoreactivity was observed for PSP in pyramidal neurons and in cells of the subgranular zone (SGZ) of the dentate gyrus (Fig. 4A). Compared to the cerebral cortex (Fig. 3) and the cerebellar cortex (Fig. 5), the overall intensity of immunostaining in the hippocampus was lower for PSP and below the limit of detection using the PSAT on tissue sections. In the pyramidal cell layer, immunolabeling of PSP was most prominent in the CA2 and CA3 regions with substantially lower expression in CA1 pyramidal cells. In addition, in the hilus of the dentate gyrus, a few PSP positive cells (Fig. 4B) co-labeled with GFAP

Fig. 5 – Expression of PSAT (A and C) and PSP (B and D) immunostaining in the adult rat cerebellum. (A) PSAT labeling of Purkinje cells. (B) PSP (red) was highly expressed in Purkinje cell bodies and did not co-localize with GFAP (green). (C) In some regions of the cerebellar cortex, PSAT was expressed in both Purkinje cells and in Bergmann glia, the latter indicated by co-labeling with anti-S100β (arrow). (D) An example of PSAT and PSP expression in the same Purkinje cell bodies and proximal dendrites. Scale bar = 50 μm.

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Fig. 6 – Expression of PSAT and PSP in postnatal day 2 rat brain. (A) Somatosensory cortex showing PSAT distribution. (B) Merged images of somatosensory cortex co-labeled with PSP and GFAP showing absence of co-localization. (C) PSAT immunostaining in the dentate gyrus of the hippocampus, and (D) merged images of PSP and GFAP in the dentate gyrus; arrowheads in D indicate examples of limited co-localization of PSP with GFAP. E, PSAT in the cerebellar cortex, and (F) merged image of PSP and GFAP in the cerebellar cortex showing absence of co-localization; arrow in F denotes an example of a PSP-labeled Purkinje neuron. H, hilus of the hippocampus. Scale bars = 50 μm.

(Fig. 4C, D), indicating some limited expression in astrocytes. The most striking observation of these experiments was the expression of PSP in the SGZ; the SGZ is a neurogenic niche in the adult brain (Cameron et al., 1998). To determine if PSP was expressed in newly born neurons, adult rat hippocampal sections were co-labeled with anti-PSP and anti-polysialated neural cell adhesion molecule (PSA-NCAM) (Figs. 4E–G). PSANCAM is a selective marker for young neurons which express the modified protein during the first 12 days of existence to aid migration (Seki and Arai, 1993). Interestingly, we observed co-

expression of PSP and PSA-NCAM in some cells within the SGZ (arrow, Figs. 4F, G). In the adult rat cerebellum, prominent expression of PSAT and PSP was observed in the cell bodies and dendritic processes of Purkinje neurons (Fig. 5). PSAT immunoreactivity was also observed in the Bergmann glia as indicated by colocalization with glial marker S-100β (Fig. 5C, white arrow). However, PSP immunoreactivity was restricted to Purkinje cells and did not co-localize with GFAP. Co-expression of PSP and PSAT was often observed in the same Purkinje cells (Fig.

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Fig. 7 – Expression of PSAT and PSP in the cerebral cortex (A, B), hippocampus (C, D) and cerebellar cortex (E, F) of the macaque monkey. Expression of PSAT (A) and PSP (B) in pyramidal cells of the cerebral cortex. The asterisks are alignment points for the insets that show higher magnification images of several pyramidal cells. (C) In hippocampus, PSAT is expressed in granule cells of the dentate gyrus (arrowhead), and in CA2/CA3 pyramidal cells (arrow). (D) PSP is expressed in the granule cells of the dentate gyrus (dg, arrowhead), cells in the hilus of the dentate gyrus (small arrow), and hippocampal CA2/CA3 pyramidal cells (large arrow). The inset in D shows a higher magnification image of immunoreactive hippocampal pyramidal cells. (E) In the cerebellar cortex, PSAT is expressed in Purkinje cells (example at arrow) but not in granule cells, nor cells in the molecular layer. F, PSP is expressed in Purkinje cells, (arrow), granule cells, (g) and small neurons (arrowhead) in the molecular layer (m). The inset shows a higher magnification image of a Purkinje cell with punctate label on the soma and apical dendrite. Scale bars for large panels A–F, 250 μm. Scale bars for insets A, B, 50 μm; D, F, 20 μm.

5D). However, examples were also observed where PSP was not expressed in the same Purkinje cells as PSAT; in the regions of the cerebellar cortex where Purkinje cells were devoid of PSAT, PSAT was expressed in Bergmann glia. It is also noteworthy that PSAT and PSP immunostaining was present in the cytosol of Purkinje cell bodies and that the cell nuclei were devoid of immunoreactivity. Together these results indicate that in the rat cerebellum, PSAT is expressed in both Bergmann glia and Purkinje neurons, while PSP expression is restricted to Purkinje neurons. Immunocytochemical analyses were also carried out on postnatal day 2 rat brains (Fig. 6). Compared to adult cortex, the cerebral cortex of 2-day-old animals showed a higher level of expression of PSAT (Fig. 6A) and particularly PSP (Fig. 6B) in most neurons throughout all cellular layers. No co-localization of PSP with GFAP was seen, indicating neuronal expression

(Fig. 6B). In the hippocampus, PSAT (Fig. 6C) and PSP immunolabeling was observed in the dentate gyrus granule neurons and in pyramidal neurons. Double labeling with PSP and GFAP showed some co-localization in the dentate gyrus (Fig. 6D), reminiscent of the co-localization observed in the adult rat hippocampus. In the postnatal day 2 cerebellum, PSAT staining was distributed throughout the cerebellar cortex (Fig. 6E), while PSP intensely stained immature Purkinje cells. No co-localization of PSAT or PSP (Fig. 6F) with GFAP was observed. Fig. 7 illustrates immunoperoxidase staining in the cerebellum, hippocampus, and cerebral cortex of the adult macaque monkey brain. In the cerebral cortex, the somas and proximal dendrites of pyramidal cells throughout the full thickness of cortex were labeled with both the PSAT (Fig. 7A) and PSP antibodies (Fig. 7B). Cell nuclei were not labeled. In the

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hippocampus, the granular layer of the dentate gyrus (dg; arrowheads in Figs. 7C, D) was darkly labeled for both PSAT (Fig. 7C) and PSP (Fig. 7D), as were somas of pyramidal cells in the CA3 region (large arrows in Figs. 7C and D and inset if Fig. 7D). Nuclei were not labeled. A few neurons in the hilus of the dentate gyrus were also labeled (Fig. 7D, small arrow). Thus overall, the pattern of PSAT and PSP expression in the adult macaque cerebral cortex and hippocampus appeared similar to that observed in the adult rat brain, although the level of PSAT expression in the macaque hippocampus was higher than that seen in the rat hippocampus. In the monkey cerebellar cortex, both the PSAT (Fig. 7E) and PSP (Fig. 7F) antibodies labeled Purkinje cell somas and the proximal portion of the dendritic trees. With PSP, the label around Purkinje cells appeared punctate (Fig. 7F, inset). However, the labeling seen with the PSAT and PSP antibodies in the cerebellum was not identical. With PSP, but not PSAT, there was also labeling of neurons in the granular layer (Fig. 7F), and in the deep cerebellar nuclei where large multipolar neurons were labeled with PSP but not with PSAT (data not shown).

3.

Discussion

We developed a novel analytical procedure for quantifying LSOP in brain tissue. Our results demonstrate that L-SOP is present at about 5 μM in rat whole brain (equivalent to approximately 1 μg/g tissue). Two previous studies (McIlwain and Bachelard, 1971; Goodnough et al., 1995), although finding levels that differ considerably from one another, have reported L-SOP concentrations in brain many times higher than those found by us. Although the reason for this discrepancy is not known, it is possible that other substances co-eluted with L-SOP in the previously reported analytical procedures. We tested various derivatizing reagents and HPLC separation techniques and found interfering peaks under several conditions. Brain levels of L-SOP found using the current HPLC method, which succeeded in resolving the interfering peaks, were confirmed using a combined HPLC/ mass spectrometry procedure (Baker et al., manuscript in preparation). Our results are much more similar to those of Kataoka et al. (1991) who reported L-SOP levels of 0.33, 0.11 and 0.35 μg/g in brain tissue from pigs, mice, and chickens, respectively. Despite the relatively low concentration of LSOP in rat whole brain, the substantially higher potency of LSOP compared to the putative endogenous agonist L-glutamate at mGluR4, suggests that L-SOP could activate these receptors in vivo (see further discussion below). Previous studies have suggested that the metabolite of L-SOP, L-serine is principally synthesized in glial cells (Savoca et al., 1995; Verleysdonk and Hamprecht, 2000). However, we found that PSAT and PSP are expressed in both neurons and in glia. The lack of induction of PSAT and PSP after kainic acidinduced seizures and gliosis indicated a primarily neuronal localization in the hippocampus (unpublished observation). Although the immunocytochemical results for PSP showed some immunolabeling of GFAP-positive astrocytes in the rat dentate gyrus (Fig. 4), more extensive immunostaining for PSP was seen in the pyramidal cells.

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Interestingly, PSP was also expressed in the subgranular zone of the hippocampus and partially co-localized with PSANCAM, a marker for young neurons. Previous work has noted a parallel between the levels of PSP and protein synthesis, and also a direct relationship between cellular demand for L-serine and the activity of PSP (Knox et al., 1969; Fell and Snell, 1988). These findings coupled with evidence that high levels of PSAT and PSP have been reported in neoplastic cell lines and tissues (Snell, 1984; Vie et al., 2008) suggest that these enzymes play a role in regulating cell proliferation and growth by controlling the amount of available L-serine. As noted above, a sufficient supply of L-serine is crucial for the normal development of the CNS. This is dramatically illustrated in children with mutations that cripple the enzymatic activity of 3-phosphoglycerate dehydrogenase, PSAT, or PSP, where severe neurological defects become apparent within several months after birth (de Koning et al., 2003), and also in 3-phosphoglycerate dehydrogenase knockout mice which die in utero (Yoshida et al., 2004). The prominent neuronal expression of PSAT and PSP was somewhat unexpected in light of reports that the upstream enzyme, 3-phosphoglycerate dehydrogenase, is mainly present in astrocytes (Furuya et al., 2000; Furuya and Watanabe, 2003) and is up-regulated in glial cells in response to kainic acid-induced seizures (Jeon et al., 2008). However, a previous study employing Northern blot analyses of glial and neuronal cultures revealed that PSP mRNA was enriched in neuronal cultures, while PSAT mRNA was expressed equally in both glial and neuronal cultures; as expected, mRNA encoding 3phosphoglycerate dehydrogenase was enriched in glial cultures (Shimizu et al., 2004). Further support for neuronal localization of PSAT and PSP also comes from a study in which an antibody to L-serine showed robust immunolabeling in both glia and in neurons, including pyramidal neurons of the cerebral cortex (Yasuda et al., 2001). Immunocytochemical analyses of the anatomical distributions in the cerebral cortex, hippocampus, and cerebellum indicated that the two enzymes appear to be generally conserved in rats and primates, and that PSAT, and especially PSP, are expressed more ubiquitously in the immature rat brain compared to the adult CNS. This is particularly true of cerebral cortical neurons where in the immunocytochemical experiments, more intense expression was seen in 2-day-old rats compared to adult rats. In the cerebral cortex of the postnatal day 2 rat, virtually every cell displayed intense PSAT and PSP immunostaining, whereas in the adult rat cortex, the number of cells expressing both enzymes was more restricted. However, in the adult rat and monkey cerebral cortex, those neurons that continued to express PSAT and PSP, appeared to express the two enzymes at relatively high levels. Immunostaining in the cerebellum displayed a convergence in Purkinje neurons where both PSAT and PSP displayed relatively high expression. PSAT expression was also high in the Bergmann glia, whereas PSP was not detected in these cells. 3-Phosphoglycerate dehydrogenase was reported to be present in Bergmann glia but not in Purkinje cells (Furuya et al., 2000; Furuya and Watanabe, 2003). Thus, the findings presented here together with previous studies indicate that Bergmann glia contain both 3-phosphoglycerate dehydrogenase and PSAT, whereas Purkinje cells possess PSAT and PSP but not 3-phosphoglycerate dehydrogenase. It is conceivable

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that phosphohydroxypyruvate, the reaction product of 3phosphoglycerate dehydrogenase, is generated in the Bergmann glia and then either converted to L-SOP by PSAT, or taken up by neighboring Purkinje cells by an as yet unidentified mechanism, and then further metabolized to L-SOP and L-serine. In this scenario, Purkinje neurons could utilize Lserine internally, and/or release it to support the growth of neighboring cells. Previously, the enzymes in the L-serine synthesis pathway were thought to be restricted to glial cells in order to relieve the metabolic burden of serine synthesis from neurons. Our findings challenge this view and indicate that L-serine is also synthesized in neurons. We highlight our observations that PSAT and PSP are present in Purkinje cells that are post-synaptic to the terminals of the cerebellar parallel fibers, and that PSAT is also expressed in the Bergmann glia. Interestingly, the parallel fiber terminals which make synaptic contacts with Purkinje cell dendrites in the molecular layer of the cerebellar cortex express the highest levels of the mGluR4 subtype of metabotropic glutamate receptor in the CNS (Kinoshita et al., 1996; Thomsen and Hampson, 1999; Corti et al., 2002). We speculate that L-SOP generated by PSAT but not immediately metabolized to L-serine by PSP, could be released from Purkinje cell dendrites that extend into the molecular layer. This release could stimulate, in a retrograde fashion, presynaptic mGluR4 receptors present on the parallel fiber terminals. The Bergmann glia whose fibers also extend into the molecular layer and envelop parallel fiber-Purkinje cell synapses (Tzingounis and Wadiche, 2007) may also release L-SOP since they display robust PSAT expression but little if any PSP. Activation of mGluR4, presumably by glutamate, is known to elicit a reduction in glutamate release at the parallel fiberPurkinje cell synapse (Pekhletski et al., 1996), and at other central synapses (Gereau and Conn, 1995; Conn and Pin, 1997). However, the much higher affinity of L-SOP for mGluR4 compared to L-glutamate indicates that mGluR4 might be preferentially activated by L-SOP. Together, our findings raise the intriguing possibility that L-SOP might act as an endogenous ligand or co-ligand (with L-glutamate) at mGluR4 in the cerebellum, and possibly at other synapses in the CNS that express mGluR4 or other Group III metabotropic glutamate receptors.

inoculated into 600 ml of M9 medium (1:30) and aerobically grown at 37 °C until the OD600 reached 0.5–0.6. The culture was maintained on ice for 15 min., then 0.5 mM isopropylthiogalactoside was added; after 20 h of incubation at 37 °C, the cells were harvested by centrifugation. For generation of mammalian expression constructs, total RNA was extracted and purified from rat forebrain using the RNeasy kit (Qiagen) and then reverse transcribed with Superscript II reverse transcriptase (Invitrogen). PSAT and PSP were cloned from the resulting cDNA using primers for PSAT (F: 5′-GCGGATCCATGGAAGCCACCAAGCAAGTG-3′, R: 5′-GCGAATTCCCAGCTGATGCATCTCCAGAAAGT-3′) and PSP (F: 5′-GCGGATCCATGGTCTCCCACTCAGAGCTG-3′, R: 5′-GCCGAATTCTTCTTCCAGTTCTCCTAGCAG-3′). The PCR conditions were 95 °C for 10 min followed by 30 cycles of 95 °C, 30 s; 60 °C, 30 s; 72 °C, 2 min; and a final elongation at 72 °C for 10 min. using Pfu polymerase (Fermentas). The amplified bands were purified with the Purelink PCR purification kit (Invitrogen) and then subcloned into the pcDNA 3.1 His-Myc vector digested with BamHI and EcoRI. HEK-293 cells were transfected in 6-well plates with 2 μg of PSAT and PSP cDNA per well using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.

4.2. Dose–response analysis in HEK-293 cells expressing mGluR4 Cell transfections and dose–response analyses were carried out as described previously (Yao et al., 2003). Briefly, HEK-293 cells were co-transfected with the cDNAs for rat mGluR4 and the promiscuous G-protein subunit Gα15 to switch signal transduction from inhibition of adenylyl cyclase to stimulation of phospholipase C; this method has been previously validated by Gomeza et al., 1996. At 16 h post-transfection the cells were sub-cultured into 96 black well plates and at 44 h post-transfection the cells were loaded with the calciumsensitive dye Fluo-4, washed, and exposed to various concentrations of the mGluR4 agonists. The fluorescence intensities were measured and quantified on a FLEXstation scanning fluorometer (Molecular Devices Corp.). The data were analyzed using the GraphPad Prism software.

4.3.

4.

Experimental procedures

4.1.

cDNA constructs

The rat c-myc tagged mGluR4 cDNA was generated and characterized as described previously (Hampson et al., 1999). The rat PSP cDNA (NCBI accession number, NM_001009679) was obtained by RT-PCR using total RNA from cultured brain cells as the template. The forward primer (5′-CGCGGCAGCCATATGGTCTCCCACTCAGAGCTG-3′) contained an NdeI site and the reverse primer (5′-GCCGGATCCTCATTCTTCCAGTTCTCCTAGCAG-3′) contained a BamHI site (restriction sites are underlined). The PCR product was digested with NdeI/BamHI and ligated into the expression vector pET3a (New England Biolabs, Boston, USA). BL21 (DE3) Gold pLysS cells were transformed with the plasmid and a single colony was cultured in LB medium at 37 °C overnight. The overnight cultured cells were

Quantitation of L-SOP in rat brain

Levels of L-SOP in adult male Sprague–Dawley (275–325 g) rat brains were measured using a modification of an HPLC procedure originally developed (Grant et al., 2006) for simultaneous analysis of D- and L-serine in plasma samples (Baker et al., submitted for publication). Briefly, the rats were euthanized by guillotine decapitation and the brains dissected out, frozen immediately in isopentane on solid carbon dioxide, then removed to another set of receptacles and stored at −80 degrees until the time of analysis. The rat handling procedures were approved by an animal ethics committee at the University of Alberta. Briefly, the analytical method involved reaction of the supernatant from a brain homogenate in methanol with a derivatizing reagent consisting of a mixture of N-isobutyryl-Lcysteine and o-phthaldialdehyde, separation of the derivatized L-SOP from other components in a Waters Alliance HPLC system and detection using a fluorescence detector.

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4.4. Generation and purification of polyclonal anti-PSP antisera A 15-residue peptide, CRLALIQPSRDQVQR, corresponding to a contiguous sequence in the rat PSP protein, plus an additional cysteine residue on the N-terminus for coupling, was covalently coupled to activated-maleimide keyhole limpet hemocyanin (Imject mcKLH, Pierce, Rockford, IL, USA). An NCBI database search indicated that this PSP peptide had no homology with any mammalian protein other than PSP. The conjugated peptide was emulsified with Freund's complete adjuvant (incomplete adjuvant was used for subsequent injections) and injected subcutaneously into two rabbits. The rabbits were boosted twice over the course of six weeks before the first titer check, and then once every 3 weeks for 2 additional months. After initial screening of the two crude antisera on western blots, one of the antisera was affinity purified using the PSP peptide immobilized to SulfoLink gel matrix (SulfoLink kit cat. #44895, Pierce).

4.5.

Western blotting

In the experiments using rat tissues, all experimental procedures were approved by the Animal Care Committee at the University of Toronto. The brains and peripheral organs were harvested from euthanized (via sodium pentobarbitol overdose) adult male Sprague–Dawley rats and postnatal day 2 rats, and homogenized with a glass-Teflon homogenizer in 50 mM Tris–HCl buffer (pH 7.4) containing 1% SDS w/v; the total protein concentration was adjusted to 1.5 mg/ml. The homogenates were prepared for electrophoresis by adding 4× sample buffer (Tris-Glycine, 2% SDS (w/v), 100 mM dithiothreitol) to a final total protein concentration of 1 μg/μl as determined by the BCA assay (Sigma, Oakville, Canada). Eight μg of total protein were loaded per well and electrophoresis was carried out using 12% Tris–HCl polyacrylamide gels. The separated proteins were electrotransferred to nitrocellulose membranes (Pall, Pensacola, FL, USA) and blocked in 5% nonfat milk protein. The membranes were incubated for 2 h at 25 °C with the primary antibodies that included the affinity purified anti-PSP rabbit polyclonal described above (2 μg/ml), or an anti-PSAT polyclonal antibody (diluted 1:1,000 - 1:4,000) raised in a chicken against amino acids 290–340 of the mammalian Psat protein (Genway Biosciences, San Diego, CA, USA). Anti-GAPDH (mouse monoclonal from SigmaAldrich Canada, Oakville, Ontario) was used as the sample loading control. The membranes were washed and incubated for 2 h with HRP-conjugated anti-rabbit antibody 1:30,000 (Pierce, Rockford, IL, USA) for the PSP antibody, or with an antichicken antibody 1:10,000 (Jackson ImmunoResearch, West Grove, PA, USA) for the PSAT antibody. After washing the membranes, the proteins were visualized using enhanced chemiluminescence detection (Amersham, Piscataway, NJ).

4.6.

Immunocytochemistry

All experimental procedures conducted on rats were approved by the Animal Care and Use Committee of the University of Toronto. For immunocytochemistry in rats, adult Sprague– Dawley rats (200–250 g) were anaesthetized by intraperitoneal

11

injection of a lethal dose of sodium pentobarbital and perfused transcardially with 0.12 M phosphate buffer (pH 7.4) followed by 4% paraformaldehyde. Brains were post-fixed in 4% paraformaldehyde overnight and cryoprotected by infiltration with 30% sucrose in phosphate-buffered saline (PBS) at 4 °C overnight. Following embedding of tissue in OCT compound (Sakura Finetek USA, Inc., Torrance, CA), 14 μm thick sagittal sections were cut on a Leica CM3050 S cryostat. The sections were mounted on poly-L-lysine-coated glass slides, air-dried overnight at 4 °C and then re-hydrated in PBS, pH 7.4. The sections were incubated in blocking buffer (0.3% Tween-20, 5% goat serum in PBS) for 60 min at 25 °C. Primary antisera were applied to the sections in a humid chamber overnight at 4 °C. The primary antisera used were: anti-PSP used at 1:100, anti-PSAT (1:1000), anti-GFAP mouse monoclonal (1:1000; Chemicon, Temecula, CA, USA), anti-MAP2 (1:200; SigmaAldrich Canada Ltd. Oakville, ON, Canada), anti-S100β 1:200 (Sigma-Aldrich Canada), and anti-PSA-NCAM (1:200; Chemicon, Temecula, CA, USA). The secondary antisera used were AlexaFluor 546 (1:1000), AlexaFluor 488 (1:1000), (Molecular Probes, Invitrogen, Carlsbad, CA, USA), Texas Red dyeconjugated rabbit anti-chicken (1:400) and FITC conjugated goat anti-chicken (1:50) from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). Where indicated DAPI (Molecular Probes, Invitrogen, Carlsbad, CA, USA) was used at a final concentration of 300 nM. The sections were then washed in PBS, cover-slipped with ProLong Gold anti-fade reagent and visualized using a Nikon E1000 fluorescent microscope, or a Zeiss LSM 510 confocal microscope equipped with the following Zeiss lenses: 20×, numerical aperture 0.75; 40×, numerical aperture 1.30 oil immersion and the appropriate filters for TRITC (anti-rabbit and anti-chicken antibodies) and FITC (anti-mouse antibody). Images were adjusted for brightness and contrast using Adobe Photoshop CS3. In the experiments using monkey brain tissue, all experimental procedures were approved by the Institutional Care and Use Committee of the University at Buffalo. An adult macaque monkey was deeply anesthetized with intravenous sodium pentobarbital, and perfused through the aorta with 0.9% saline followed by 4% paraformaldehyde in 0.1 M phosphate-buffered saline (PBS). The brain was removed and divided into two blocks that were cut in the frontal plane. The tissue blocks were then cryoprotected in graded solutions (15% then 30%) of sucrose in PBS, frozen, and stored at -70 °C in a cryoprotection solution of 30% ethylene glycol, 30% glycerol in PBS. Frozen frontal sections 50 μm thick were cut on a sliding microtome and the sections were stored in a cryoprotectant solution of glycerol-ethylene glycol at −20 °C. For immunocytochemistry, sections were rinsed in PBS (all rinses were 3 × 10 min at 26 °C, with gentle agitation), blocked for 30 min in PBS, 1% with Triton-X, 1% bovine serum albumin, and 1.5% normal goat serum (Vector Laboratories), then incubated at 4 °C overnight with the primary antibodies (PSAT, 1:000; antiPSP 1:500). Subsequent processing was with a Vector ABC Elite kit following manufacturer's instructions, using Vector biotinylated secondary antibodies (anti-rabbit for PSP and antichicken for PSAT). Immunoreactivity was visualized with a glucose oxidase modification of a 3,3′ diaminobenzidine protocol (Shu et al., 1988). Control sections were processed identically except for omission of the primary antibodies.

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Acknowledgments We thank Drs. Jeffrey Henderson helpful advice, Dr. David Bender for the gift of the macaque monkey tissue, and Ms. Gail Rauw for expert technical assistance. This work was supported by the Canadian Institutes for Health Research (via an operating grant to DRH and GBB) and by an Ontario Graduate Scholarship (to JEA).

REFERENCES

Baek, J.Y., Jun, D.Y., Taub, D., Kim, Y.H., 2003. Characterization of human phosphoserine aminotransferase involved in the phosphorylated pathway of L-serine biosynthesis. Biochem. J. 373, 191–200. Brauner-Osborne, H., Wellendorph, P., Jensen, A.A., 2007. Structure, pharmacology and therapeutic prospects of family C G-protein coupled receptors. Curr. Drug Targets 8, 169–184. Cameron, H.A., Tanapat, P., Gould, E., 1998. Adrenal steroids and N-methyl-D-aspartate receptor activation regulate neurogenesis in the dentate gyrus of adult rats through a common pathway. Neuroscience 82, 349–354. Collet, J.F., Stroobant, V., Van, S.E., 1999. Mechanistic studies of phosphoserine phosphatase, an enzyme related to P-type ATPases. J. Biol. Chem. 274, 33985–33990. Conn, P.J., Pin, J.P., 1997. Pharmacology and functions of metabotropic glutamate receptors. Annu. Rev. Pharmacol. Toxicol. 37, 205–237. Corti, C., Aldegheri, L., Somogyi, P., Ferraguti, F., 2002. Distribution and synaptic localisation of the metabotropic glutamate receptor 4 (mGluR4) in the rodent CNS. Neuroscience 110, 403–420. de Koning, T.J., Snell, K., Duran, M., Berger, R., Poll-The, BT, Surtees, R., 2003. L-Serine in disease and development. Biochem. J. 371, 653–661. Fell, D.A., Snell, K., 1988. Control analysis of mammalian serine biosynthesis. Feedback inhibition on the final step. Biochem. J. 256, 97–101. Furuya, S., Watanabe, M., 2003. Novel neuroglial and glioglial relationships mediated by L-serine metabolism. Arch. Histol. Cytol. 66, 109–121. Furuya, S., Tabata, T., Mitoma, J., Yamada, K., Yamasaki, M., Makino, A., Yamamoto, T., Watanabe, M., Kano, M., Hirabayashi, Y., 2000. L-serine and glycine serve as major astroglia-derived trophic factors for cerebellar Purkinje neurons. Proc. Natl. Acad. Sci. U. S. A. 97, 11528–11533. Gereau, R.W., Conn, P.J., 1995. Multiple presynaptic metabotropic glutamate receptors modulate excitatory and inhibitory synaptic transmission in hippocampal area CA1. J. Neurosci. 15, 6879–6889. Gomeza, J., Joly, C., Kuhn, R., Knopfel, T., Bockaert, J., Pin, J.P., 1996. The second intracellular loop of metabotropic glutamate receptor 1 cooperates with the other intracellular domains to control coupling to G-proteins. J. Biol. Chem. 271, 2199–2205. Goodnough, D.B., Lutz, M.P., Wood, P.L., 1995. Separation and quantification of D- and L-phosphoserine in rat brain using N alpha-(2,4-dinitro-5-fluorophenyl)-L-alaninamide (Marfey's reagent) by high-performance liquid chromatography with ultraviolet detection. J. Chromatogr. B Biomed. Appl. 672, 290–294. Grant, S.L., Shulman, Y., Tibbo, P., Hampson, D.R., Baker, G.B., 2006. Determination of d-serine and related neuroactive amino acids in human plasma by high-performance liquid chromatography with fluorimetric detection. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 844, 278–282.

Hampson, D.R., Huang, X.P., Pekhletski, R., Peltekova, V., Hornby, G., Thomsen, C., Thogersen, H., 1999. Probing the ligand-binding domain of the mGluR4 subtype of metabotropic glutamate receptor. J. Biol. Chem. 274, 33488–33495. Hampson, DR, Rose, EM, Antflick, JE, 2008. The structures of the metabotropic glutamate receptors. In: GereauIV IV, R.W., Swanson, G.T. (Eds.), The Glutamate Receptors. Humana Press. Hart, C.E., Race, V., Achouri, Y., Wiame, E., Sharrard, M., Olpin, S.E., Watkinson, J., Bonham, J.R., Jaeken, J., Matthijs, G., Van, S.E., 2007. Phosphoserine aminotransferase deficiency: a novel disorder of the serine biosynthesis pathway. Am. J. Hum. Genet. 80, 931–937. Hester, G., Stark, W., Moser, M., Kallen, J., Markovic-Housley, Z., Jansonius, J.N., 1999. Crystal structure of phosphoserine aminotransferase from Escherichia coli at 2.3 A resolution: comparison of the unligated enzyme and a complex with alpha-methyl-l-glutamate. J. Mol. Biol. 286, 829–850. Iacovelli, L., Arcella, A., Battaglia, G., Pazzaglia, S., Aronica, E., Spinsanti, P., Caruso, A., De, S.E., Saran, A., Gulino, A., D'Onofrio, M., Giangaspero, F., Nicoletti, F., 2006. Pharmacological activation of mGlu4 metabotropic glutamate receptors inhibits the growth of medulloblastomas. J. Neurosci. 26, 8388–8397. Jeon, G.S., Choi, D.H., Lee, H.N., Kim, D.W., Chung, C.K., Cho, S.S., 2009. Expression of L-serine biosynthetic enzyme 3-phosphoglycerate dehydrogenase (Phgdh) and neutral amino acid transporter ASCT1 following an excitotoxic lesion in the mouse hippocampus. Neurochem. Res. 34, 827–834. Kataoka, H., Sakiyama, N., Makita, M., 1991. Distribution and contents of free O-phosphoamino acids in animal tissues. J. Biochem. 109, 577–580. Kinoshita, A., Ohishi, H., Nomura, S., Shigemoto, R., Nakanishi, S., Mizuno, N., 1996. Presynaptic localization of a metabotropic glutamate receptor, mGluR4a, in the cerebellar cortex: a light and electron microscope study in the rat. Neurosci. Lett. 207, 199–202. Klunk, W.E., McClure, R.J., Pettegrew, J.W., 1991. L-phosphoserine, a metabolite elevated in Alzheimer's disease, interacts with specific L-glutamate receptor subtypes. J. Neurochem. 56, 1997–2003. Knox, W.E., Herzfeld, A., Hudson, J., 1969. Phosphoserine phosphatase distribution in normal and neoplastic rat tissues. Arch. Biochem. Biophys. 132, 397–403. McIlwain, H, Bachelard, HS, 1971. Biochemistry and the Central Nervous System. Churchill Livingstone, London, U.K. Mothet, J.P., Parent, A.T., Wolosker, H., Brady Jr., R.O., Linden, D.J., Ferris, C.D., Rogawski, M.A., Snyder, S.H., 2000. D-serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor. Proc. Natl. Acad. Sci. U. S. A. 97, 4926–4931. Nakamichi, N., Yoshida, K., Ishioka, Y., Makanga, J.O., Fukui, M., Yoneyama, M., Kitayama, T., Nakamura, N., Taniura, H., Yoneda, Y., 2008. Group III metabotropic glutamate receptor activation suppresses self-replication of undifferentiated neocortical progenitor cells. J. Neurochem. 105, 1996–2012. Nakano, I., Dougherty, J.D., Kim, K., Klement, I., Geschwind, D.H., Kornblum, H.I., 2007. Phosphoserine phosphatase is expressed in the neural stem cell niche and regulates neural stem and progenitor cell proliferation. Stem Cells 25, 1975–1984. Naples, M.A., Hampson, D.R., 2001. Pharmacological profiles of the metabotropic glutamate receptor ligands. Neuropharmacology 40, 170–177. Naur, P., Hansen, K.B., Kristensen, A.S., Dravid, S.M., Pickering, D.S., Olsen, L., Vestergaard, B., Egebjerg, J., Gajhede, M., Traynelis, S.F., Kastrup, J.S., 2007. Ionotropic glutamate-like receptor {delta}2 binds D-serine and glycine. Proc. Natl. Acad. Sci. U. S. A. 104, 14116–14121.

BR A IN RE S EA RCH 1 3 00 ( 20 0 9 ) 1 –1 3

Nicoletti, F., Wroblewski, J.T., Iadarola, M.J., Costa, E., 1986. Serine-O-phosphate, an endogenous metabolite, inhibits the stimulation of inositol phospholipid hydrolysis elicited by ibotenic acid in rat hippocampal slices. Neuropharmacology 25, 335–338. Niswender, C.M., Johnson, K.A., Weaver, C.D., Jones, C.K., Xiang, Z., Luo, Q., Rodriguez, A.L., Marlo, J.E., de, P.T., Thompson, A.D., Days, E.L., Nalywajko, T., Austin, C.A., Williams, M.B., Ayala, J.E., Williams, R., Lindsley, C.W., Conn, P.J., 2008. Discovery, characterization, and antiparkinsonian effect of novel positive allosteric modulators of metabotropic glutamate receptor 4. Mol. Pharmacol. 74, 1345–1358. Pekhletski, R., Gerlai, R., Overstreet, L.S., Huang, X.P., Agopyan, N., Slater, N.T., Abramow-Newerly, W., Roder, J.C., Hampson, D.R., 1996. Impaired cerebellar synaptic plasticity and motor performance in mice lacking the mGluR4 subtype of metabotropic glutamate receptor. J. Neurosci. 16, 6364–6373. Savoca, R., Ziegler, U., Sonderegger, P., 1995. Effects of L-serine on neurons in vitro. J. Neurosci. Methods 61, 159–167. Saxe, J.P., Wu, H., Kelly, T.K., Phelps, M.E., Sun, Y.E., Kornblum, H.I., Huang, J., 2007. A phenotypic small-molecule screen identifies an orphan ligand–receptor pair that regulates neural stem cell differentiation. Chem. Biol. 14, 1019–1030. Schoepp, D.D., Jane, D.E., Monn, J.A., 1999. Pharmacological agents acting at subtypes of metabotropic glutamate receptors. Neuropharmacology 38, 1431–1476. Seki, T., Arai, Y., 1993. Highly polysialylated neural cell adhesion molecule (NCAM-H) is expressed by newly generated granule cells in the dentate gyrus of the adult rat. J. Neurosci. 13, 2351–2358. Shimizu, M., Furuya, S., Shinoda, Y., Mitoma, J., Okamura, T., Miyoshi, I., Kasai, N., Hirabayashi, Y., Suzuki, Y., 2004. Functional analysis of mouse 3-phosphoglycerate dehydrogenase (Phgdh) gene promoter in developing brain. J. Neurosci. Res. 76, 623–632. Shleper, M., Kartvelishvily, E., Wolosker, H., 2005. D-serine is the dominant endogenous coagonist for NMDA receptor

13

neurotoxicity in organotypic hippocampal slices. J. Neurosci. 25, 9413–9417. Shu, S.Y., Ju, G., Fan, L.Z., 1988. The glucose oxidase-DAB-nickel method in peroxidase histochemistry of the nervous system. Neurosci. Lett. 85, 169–171. Snell, K., 1984. Enzymes of serine metabolism in normal, developing and neoplastic rat tissues. Adv. Enzyme Regul. 22, 325–400. Thomsen, C., Hampson, D.R., 1999. Contribution of metabotropic glutamate receptor mGluR4 to L-2-[3H]amino-4-phosphonobutyrate binding in mouse brain. J. Neurochem. 72, 835–840. Thomsen, C., Pekhletski, R., Haldeman, B., Gilbert, T.A., O'Hara, P., Hampson, D.R., 1997. Cloning and characterization of a metabotropic glutamate receptor, mGluR4b. Neuropharmacology 36, 21–30. Tzingounis, A.V., Wadiche, J.I., 2007. Glutamate transporters: confining runaway excitation by shaping synaptic transmission. Nat. Rev. Neurosci. 8, 935–947. Verleysdonk, S., Hamprecht, B., 2000. Synthesis and release of L-serine by rat astroglia-rich primary cultures. Glia 30, 19–26. Vie, N., Copois, V., Bascoul-Mollevi, C., Denis, V., Bec, N., Robert, B., Fraslon, C., Conseiller, E., Molina, F., Larroque, C., Martineau, P., Del, R.M., Gongora, C., 2008. Overexpression of phosphoserine aminotransferase PSAT1 stimulates cell growth and increases chemoresistance of colon cancer cells. Mol. Cancer 7, 14. Yao, Y., Pattabiraman, N., Michne, W.F., Huang, X.P., Hampson, D.R., 2003. Molecular modeling and mutagenesis of the ligand-binding pocket of the mGlu3 subtype of metabotropic glutamate receptor. J. Neurochem. 86, 947–957. Yasuda, E., Ma, N., Semba, R., 2001. Immunohistochemical demonstration of L-serine distribution in the rat brain. NeuroReport 12, 1027–1030. Yoshida, K., Furuya, S., Osuka, S., Mitoma, J., Shinoda, Y., Watanabe, M., Azuma, N., Tanaka, H., Hashikawa, T., Itohara, S., Hirabayashi, Y., 2004. Targeted disruption of the mouse 3-phosphoglycerate dehydrogenase gene causes severe neurodevelopmental defects and results in embryonic lethality. J. Biol. Chem. 279, 3573–3577.