Ryuichi Konno

Free d-serine, d-aspartate and d-alanine in central nervous system and serum in mutant mice lacking d-amino acid oxidase

We have examined whether D-amino acid oxidase (DAO) regulates free D-serine content using mutant ddY/DAO- mice lacking DAO activity. We find that the content of D-serine in the serum and cerebellum of mutant mice is much higher than that of normal mice, whereas a slight but significant difference in the cerebral D-serine level is observed between the two strains. These results suggest that, although DAO may participate in the catabolism of D-serine in the cerebellum and periphery, there appears to be other mechanisms for catabolism of endogenous D-serine in the brain.

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.

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.

Exaggerated responses to chronic nociceptive stimuli and enhancement of N-methyl-D-aspartate receptor-mediated synaptic transmission in mutant mice lacking D-amino-acid oxidase.

Formalin-induced nociceptive behaviors and N-methyl-D-aspartate (NMDA) subtype glutamate receptor-mediated excitatory synaptic transmission were analyzed in mutant mice lacking D-amino-acid oxidase, which catalyzes the oxidative deamination of D-amino acids. The second phase of the formalin-induced licking response, a part of which is known to be mediated by NMDA receptors in the spinal cord, was significantly augmented in mutant mice. NMDA receptor-mediated excitatory postsynaptic currents recorded from spinal cord dorsal horn neurons by tight-seal whole-cell methods were significantly potentiated in mutant mice. The present observations provide another line of evidence that D-serine functions as an endogenous coagonist at the glycine site of NMDA receptors, and raise the possibility that D-amino-acid oxidase exerts a neuromodulatory function by controlling the concentration of D-serine in the central nervous system.

Spatial learning and long-term potentiation of mutant mice lacking d-amino-acid oxidase

We evaluated the role of D-amino-acid oxidase on spatial learning and long-term potentiation (LTP) in the hippocampus, since this enzyme metabolizes D-amino-acids, some of which enhance the N-methyl-D-aspartate receptor functions. The Morris water maze learning and the LTP in the CA1 area of the hippocampal slice were observed in wild-type mice and mutant mice lacking D-amino-acid oxidase. The mutant mice showed significantly shorter platform search times in the water maze and significantly larger hippocampal LTPs than the wild-type mice. These results suggest that the abundant D-amino-acids in the mutant mouse brain facilitate hippocampal LTP and spatial learning.

The presence of free d-alanine, d-proline and d-serine in mice

The presence of free D-alanine, D-proline and D-serine was demonstrated in mammalian tissues, using a mutant mouse strain lacking D-amino acid oxidase. In the experiment, free amino acids from the kidney and serum were derivatized with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA) to diastereomers, separated by two-dimensional thin-layer chromatography (TLC), and analysed by reversed-phase high-performance liquid chromatography (HPLC) for the resolution of D- and L-isomers. D/L ratios of alanine, proline and serine were obtained based on the peak areas of HPLC.

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.

Mice lacking d-amino acid oxidase activity display marked attenuation of stereotypy and ataxia induced by MK-801

The behavioral effects produced by MK-801 (0.4 mg/kg) were compared in mutant DAO-/- mice lacking D-amino acid oxidase activity and normal DAO+/+ mice. Mutant mice display marked diminution of stereotypy and ataxia induced by MK-801 compared to normal mice. Because the D-serine level in the brain of mutant mice is significantly higher than that of normal mice, the elevated D-serine in the brain of mutant mice could antagonize MK-801-induced stereotypy and ataxia.

A single-base-pair substitution abolishes d-amino-acid oxidase activity in the mouse

Mutant ddY/DAO- mice lacking D-amino-acid oxidase (DAO) activity were examined for the cause of their lack of enzyme activity. Total RNA was extracted from the kidney of the ddY/DAO- mice and cDNA was synthesized. After cDNA encoding DAO was amplified by the polymerase chain reaction it was cloned into a plasmid and sequenced. Comparison of the DAO cDNA sequence with that of normal BALB/c mice revealed the presence of a single-base substitution (G----A) which causes a Gly-181----Arg substitution in the middle of the enzyme molecule. The mutant DAO cDNA was inserted into an expression vector and was expressed in transfected COS-1 cells. The transfected cells synthesized the DAO mutant protein, but they did not show DAO activity. In contrast, when cells were transfected with an expression vector carrying wild-type DAO cDNA, where the substituted base-pair was replaced by a normal base-pair, they showed DAO activity. These results indicate that the single base-pair substitution is the cause of the loss of DAO activity in the ddY/DAO- mice.