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, 285 (53), 41380-90

Brain-specific Phgdh Deletion Reveals a Pivotal Role for L-serine Biosynthesis in Controlling the Level of D-serine, an N-methyl-D-aspartate Receptor Co-Agonist, in Adult Brain

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Brain-specific Phgdh Deletion Reveals a Pivotal Role for L-serine Biosynthesis in Controlling the Level of D-serine, an N-methyl-D-aspartate Receptor Co-Agonist, in Adult Brain

Jung Hoon Yang et al. J Biol Chem.

Abstract

In mammalian brain, D-serine is synthesized from L-serine by serine racemase, and it functions as an obligatory co-agonist at the glycine modulatory site of N-methyl-D-aspartate (NMDA)-selective glutamate receptors. Although diminution in D-serine level has been implicated in NMDA receptor hypofunction, which is thought to occur in schizophrenia, the source of the precursor L-serine and its role in D-serine metabolism in adult brain have yet to be determined. We investigated whether L-serine synthesized in brain via the phosphorylated pathway is essential for D-serine synthesis by generating mice with a conditional deletion of D-3-phosphoglycerate dehydrogenase (Phgdh; EC 1.1.1.95). This enzyme catalyzes the first step in L-serine synthesis via the phosphorylated pathway. HPLC analysis of serine enantiomers demonstrated that both L- and D-serine levels were markedly decreased in the cerebral cortex and hippocampus of conditional knock-out mice, whereas the serine deficiency did not alter protein expression levels of serine racemase and NMDA receptor subunits in these regions. The present study provides definitive proof that L-serine-synthesized endogenously via the phosphorylated pathway is a key rate-limiting factor for maintaining steady-state levels of D-serine in adult brain. Furthermore, NMDA-evoked transcription of Arc, an immediate early gene, was diminished in the hippocampus of conditional knock-out mice. Thus, this study demonstrates that in mature neuronal circuits L-serine availability determines the rate of D-serine synthesis in the forebrain and controls NMDA receptor function at least in the hippocampus.

Figures

FIGURE 1.
FIGURE 1.
Targeted inactivation of Phgdh in brain. A, shown is a schematic representation of the wild-type Phgdh allele, targeting vector, targeted allele, and conditional allele. Exons 3–5 are shown as solid boxes, loxP sequences are shown as solid triangles, and the arrowheads indicate the locations of the PCR primers used for genotyping. Open boxes represent the neowt and DT-A gene cassettes used for positive and negative selection, respectively. The conditional allele was generated by partial excision of the targeted allele using EIIa-Cre deleter mice as reported (40, 41). Restriction enzyme map: Nh, NheI; P, PstI; S, SacI; H, HindIII; E, EcoRV; Sp, SphI; N, NsiI. B, genotyping PCR from Phgdh+/+, Phgdhflox/+, and Phgdhflox/flox mice with or without the hGFAP-Cre transgene (Cre) is shown. C, shown is a Western blot analysis of Phgdh (57 kDa) in protein lysates prepared from the cerebral cortex (upper) and hippocampus (lower) of littermate control (Phgdhflox/flox, Floxed) and conditional mutant (hGFAP+/Cre::Phgdhflox/flox, CKO) mice at postnatal day 56. Comparable staining of Gapdh was used to verify equivalent protein loading.
FIGURE 2.
FIGURE 2.
Brain-specific deletion of Phgdh causes postnatal microcephaly. A, shown is the dorsal view of CKO (left) and Floxed (right) brain at postnatal day 0. Bar, 1 mm. B, coronal sections are shown of CKO (left) and Floxed (right) forebrain region with hematoxylin and eosin staining at postnatal day 0. Bar, 1 mm. C, shown is quantification of brain weight. Mean brain weight of newborn male pups (n = 5) is shown. Note that the gross morphology and weight of brain of CKO mice are indistinguishable from Floxed littermates (right). D, shown is a dorsal view of CKO (left) and Floxed (right) brain at postnatal day 42. E, shown are coronal sections of CKO (left) and Floxed (right) forebrain region with hematoxylin and eosin staining at postnatal day 42. Bar, 1 mm. The brain of a Phgdh CKO mouse is consistently smaller than its Floxed littermate. The arrows indicate the length of forebrain. F, quantification of brain weight at age 6 weeks (male, n = 6) is shown.
FIGURE 3.
FIGURE 3.
l- and d-Serine levels in CKO serum and brain. A, serum l-serine concentrations in Floxed and CKO mice are shown. B, serum d-serine concentrations in Floxed and CKO mice are shown. C, serum glycine concentrations in Floxed and CKO mice are shown. D, l- (L) and d-serine (D) levels in the cerebral cortex of Floxed and CKO mice are shown. E, glycine content in the cerebral cortex of Floxed and CKO mice are shown. F, l- and d-serine levels in the hippocampus of Floxed and CKO mice are shown. G, glycine content in the hippocampus of Floxed and CKO mice are shown. Data represent the mean ± S.E. (Floxed: n = 7, CKO: n = 6). Differences between genotypes were analyzed by Student's t test (see “Experimental Procedures”).
FIGURE 4.
FIGURE 4.
Changes in l- and d-serine levels in the cerebral cortex after l-serine administration. A–D, shown is the l-serine (A and C) and d-serine (B and D) content in the cerebral cortex of CKO (A and B) and Floxed (C and D) mice 3 h after intraperitoneal injection of excess l-serine. Data represent the mean ± S.E. E, shown is the ratio of d-serine to total serine in Floxed and CKO cortex after serine administration. Data represent the mean ± S.E. NT, no treatment, n = 7 for Floxed, n = 6 for CKO; Saline, saline-injected, n = 3; l-Ser, l-serine-injected, n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.005 versus saline-injected mice (one-way analysis of variance, Dunnett's post-hoc test). The l- and d-serine data shown in Fig. 3D were used as no treatment in A–D. In E, ***, p < 0.005 versus saline-injected Floxed mice (one-way analysis of variance, Dunnett's post-hoc test).
FIGURE 5.
FIGURE 5.
Expression of serine racemase and NMDA receptor subunits. A and B, Srr protein was detected in protein lysates (10 μg) of the cerebral cortex (A) and hippocampus (B) by Western blotting. Srr protein levels were normalized to Gapdh protein levels. Data represent the mean ± S.E. (cerebral cortex: n = 5, hippocampus: n = 4). C and D, NMDA receptor subunits NR1 and NR2B were detected in protein lysates (10 μg) of the cerebral cortex (C) and hippocampus (D) by Western blotting. Expression levels were normalized to Gapdh levels. Data represent the mean ± S.E. (cerebral cortex, n = 5; hippocampus, n = 4).
FIGURE 6.
FIGURE 6.
Expression of neuronal and astroglial marker proteins. A and B, neuronal markers SNAP-25 and NSE were detected in protein lysates (10 μg) of the cerebral cortex (A) and hippocampus (B) by Western blotting. Expression levels were normalized to Gapdh levels. Data represent the mean ± S.E. (cerebral cortex, n = 5; hippocampus, n = 4). C and D, Gfap was detected in protein lysates (10 μg) of the cerebral cortex (C) and hippocampus (D) by Western blotting. Expression levels were normalized to Gapdh levels. Data represent the mean ± S.E. (cerebral cortex, n = 5; hippocampus: n = 4).
FIGURE 7.
FIGURE 7.
Altered Arc induction in hippocampus evoked by NMDA administration. Arc mRNA levels 3 h after administration of NMDA or PBS in the hippocampus of wild-type (A), Floxed (B), and CKO (B) mice were measured with quantitative real-time PCR. Expression levels of Arc mRNA are shown as arbitrary units normalized to Gapdh levels. Data represent the mean ± S.E. (wild type, n = 4 for each treatment; Floxed, n = 5 for PBS treatment, n = 6 for NMDA treatment; CKO, n = 3 for PBS treatment, n = 5 for NMDA treatment). Differences between treatments were analyzed by Student's t test (A) or one-way analysis of variance, Dunnett's post-hoc test (B) (*, p < 0.05; **, p < 0.005).

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