Kenji Hamase

Regional distribution and postnatal changes of d-amino acids in rat brain

Regional distribution of D-amino acids in rat brain was studied by the modified highly sensitive analytical method which was previously developed. The method includes fluorogenic derivatization of each amino acid, isolation of each amino acid by reverse-phase HPLC, followed by enantiomeric separation with Pirkle-type chiral stationary phases. D-Amino acid contents were determined in the cerebrum, cerebellum, hippocampus, medulla oblongata, pituitary gland and pineal gland. D-Aspartic acid was observed in the pineal gland (3524 +/- 263 nmol/g, data are for male rats of 6 weeks of age) and the pituitary gland (80.5 +/- 9.0 nmol/g). D-Serine was found in various regions of the brain except for the cerebellum and medulla oblongata. D-Alanine was observed exclusively in the pituitary gland (25.9 +/- 4.4 nmol/g), whereas D-leucine was found in the pineal gland (3.4 +/- 0.4 nmol/g) and the hippocampus (1.6 +/- 0.07 nmol/g). No other D-amino acids were detected in the brain. The contents of D-aspartic acid in the pituitary gland and D-serine in the pineal gland were higher in female rats. In contrast the contents of D-alanine in the pituitary gland and D-leucine in the pineal gland and the hippocampus were higher in males. Postnatal changes of D-aspartic acid and D-leucine in the pineal gland and D-alanine in the pituitary gland were also investigated. The results described in this paper suggested that distinct regulatory mechanisms exist for individual D-amino acids in the corresponding region of rat brain.

D-Amino acids in mammals and their diagnostic value

Substantial amounts of D-amino acids are present in mammalian tissues; their function, origin and relationship between pathophysiological processes have been of great interest over the last two decades. In the present article, analytical methods including chromatographic, electrophoretic and enzymatic methods to determine D-amino acids in mammalian tissues are reviewed, and the distribution of these D-amino acids in mammals is discussed. An overview of the function, origin and relationship between the amino acids and pathophysiological processes is also given.

Determination of free d-aspartic acid, d-serine and d-alanine in the brain of mutant mice lacking d-amino-acid oxidase activity

A simple and precise method for the simultaneous determination of free D-aspartic acid, D-serine and D-alanine in mouse brain tissues was established, using a reversed-phase HPLC system with widely used pre-column derivatizing reagents, o-phthaldialdehyde and N-t-butyloxycarbonyl-L-cysteine. With the present method, the contents of these three D-amino acids in hippocampus, hypothalamus, pituitary gland, pineal gland and medulla oblongata as well as cerebrum and cerebellum of mutant mice lacking D-amino-acid oxidase activity were determined and compared with those obtained for control mice. In both mice, extremely high contents of D-serine were observed in forebrain (100-400 nmol/g wet tissue), and the contents were small in pituitary and pineal glands. While, D-serine contents in cerebellum and medulla oblongata of mutant mice were about ten times higher than those in control mice. In contrast, D-alanine contents in mutant mice were higher than those in control mice in all brain regions and serum.

d-Amino acid oxidase controls motoneuron degeneration through d-serine

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder involving an extensive loss of motoneurons. Aberrant excitability of motoneurons has been implicated in the pathogenesis of selective motoneuronal death in ALS. d-Serine, an endogenous coagonist of N-methyl-d-aspartate receptors, exacerbates motoneuronal death and is increased both in patients with sporadic/familial ALS and in a G93A-SOD1 mouse model of ALS (mSOD1 mouse). More recently, a unique mutation in the d-amino acid oxidase (DAO) gene, encoding a d-serine degrading enzyme, was reported to be associated with classical familial ALS. However, whether DAO affects the motoneuronal phenotype and d-serine increase in ALS remains uncertain. Here, we show that genetic inactivation of DAO in mice reduces the number and size of lower motoneurons with axonal degeneration, and that suppressed DAO activity in reactive astrocytes in the reticulospinal tract, one of the major inputs to the lower motoneurons, predominantly contributes to the d-serine increase in the mSOD1 mouse. The DAO inactivity resulted from expressional down-regulation, which was reversed by inhibitors of a glutamate receptor and MEK, but not by those of inflammatory stimuli. Our findings provide evidence that DAO has a pivotal role in motoneuron degeneration through d-serine regulation and that inactivity of DAO is a common feature between the mSOD1 ALS mouse model and the mutant DAO-associated familial ALS. The therapeutic benefit of reducing d-serine or controlling DAO activity in ALS should be tested in future studies.

ANALYTICAL CHEMISTRY AND BIOCHEMISTRY OF D-AMINO ACIDS

The methodologies for the analysis of D-amino acids in biological materials have been reviewed, including the use of enzymes, gas and liquid chromatography with chiral stationary phases and diastereomer derivatization with chiral reagents followed by GC or HPLC separation. The distribution of D-amino acids in the body, their origin, metabolism and possible roles in human diseases are discussed.

D -Serine regulates cerebellar LTD and motor coordination through the δ2 glutamate receptor

D-Serine (D-Ser) is an endogenous co-agonist for NMDA receptors and regulates neurotransmission and synaptic plasticity in the forebrain. D-Ser is also found in the cerebellum during the early postnatal period. Although D-Ser binds to the δ2 glutamate receptor (GluD2, Grid2) in vitro, its physiological significance has remained unclear. Here we show that D-Ser serves as an endogenous ligand for GluD2 to regulate long-term depression (LTD) at synapses between parallel fibers and Purkinje cells in the immature cerebellum. D-Ser was released mainly from Bergmann glia after the burst stimulation of parallel fibers in immature, but not mature, cerebellum. D-Ser rapidly induced endocytosis of AMPA receptors and mutually occluded LTD in wild-type, but not Grid2-null, Purkinje cells. Moreover, mice expressing mutant GluD2 in which the binding site for D-Ser was disrupted showed impaired LTD and motor dyscoordination during development. These results indicate that glial D-Ser regulates synaptic plasticity and cerebellar functions by interacting with GluD2.

Determination of d-alanine in the rat central nervous system and periphery using column-switching high-performance liquid chromatography

A column-switching chiral HPLC system for the determination of minute amounts of D-Ala in mammalian tissues has been established. D-Ala and its L-enantiomer are purified as a DL mixture on a micro-ODS column after precolumn fluorescence derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and are introduced to a chiral column to determine each enantiomer. The calibration curve of D-Ala spiked into a rat cerebellum sample is linear from 5 to 5000 fmol with a correlation coefficient of 1.0000. The lower limit of quantitation of D-Ala is 5 fmol (S/N=5). Within-day and day-to-day precisions of spiked D-Ala (15 fmol) are 3.9 and 4.8% (R.S.D), respectively. With this system, the anatomical distribution of free D-Ala in the rat central nervous system and periphery has been investigated. Among the 22 examined tissues of the rat, the highest amount of D-Ala has been observed in the anterior pituitary gland (86.4+/-9.9 nmol/g wet tissue), and the second highest amount has been observed in the pancreas (29.2+/-5.0 nmol/g wet tissue). Postnatal and day-night changes in D-Ala amounts in the anterior pituitary gland have also been studied. The amount of D-Ala is highest at 6 weeks of age and significantly decreases with age, and the amount of D-Ala is significantly higher during the daytime than during the nighttime.

Determination of d-serine and d-alanine in the tissues and physiological fluids of mice with various d-amino-acid oxidase activities using two-dimensional high-performance liquid chromatography with fluorescence detection

Two-dimensional-HPLC procedures have been established for the sensitive and selective determination of D-serine (D-Ser) and D-alanine (D-Ala), and their amounts in the tissues and physiological fluids of mice with various D-amino-acid oxidase (DAO) activities have been demonstrated. These two D-amino acids are modulators of the N-methyl-D-aspartate receptor mediated neurotransmission, and the alterations in their amounts following the changes in the DAO activity are matters of interest. After pre-column derivatization with 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F), the D-amino acids were determined by the 2D-HPLC system with fluorescence detectors. As the first dimension, a microbore-monolithic-ODS column (750 mm x 0.53 mm I.D.) was adopted and a self-packed narrowbore-Pirkle type enantioselective column (Sumichiral OA-2500S, 250 mm x 1.5 mm I.D.) was selected for the second dimension. The lower limits of quantitation of D-Ser and D-Ala were 500 amol, and the within-day and day-to-day precisions were less than 6.8%. Using these methods, the amounts of D-Ser and D-Ala in 6 brain tissues, 4 peripheral tissues, serum and urine of mice having various DAO activities were determined; the amounts of these D-amino acids were drastically increased with a lowering of the DAO activity except for the cases of D-Ser in the frontal brain regions. The present micro-2D-HPLC procedures are powerful tools for the determination of small amounts of D-Ser and D-Ala in mammalian samples, and the obtained results would be useful for developing novel drugs that modulate the DAO activity, such as DAO inhibitors, against neuronal diseases.

HPLC analysis of naturally occurring free D-amino acids in mammals.

D-amino acids are currently recognized as naturally occurring physiologically active substances and biomarkers in mammals. The progress of analytical technologies, mostly high resolution chromatographic or electrodriven separation methods, has significantly contributed to the advances in D-amino acid research in real biological matrices. In this review, we would like to describe the D-amino acid research, from the discovery of appreciable amounts of free D-amino acids in mammals to the current metabolomics study focusing on amino acid enantiomers. The liquid phase enantioselective analytical methods utilized for the determination of D-amino acids in mammals including human beings will be discussed.

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

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.