Lacking D-amino Acid Oxidase Activity Research Articles

D-amino acid oxidase contributes IgA nephropathy through regulation of IgA production in intestinal mucosa

Abstract Gut mucosa protects our body against threats of bacteria using antimicrobial proteins such as defensins, cathelicidins or lysozyme. D-amino acid oxidase (DAO) is one of the antimicrobial protein that is released from goblet cells into lumen and catabolize D-amino acids, which is essential for almost all bacteria, into antibacterial agent; H2O2. IgA is major immunoglobulin in gut. Maturation of B cells into IgA-secreting plasma cells are lead through two pathways; T cell dependent or independent mechanism. A number of reports indicated the accumulation of systemic IgA in the animals lacking antimicrobial proteins. However, the mechanism how loss of innate immunity affects acquired immunity is yet to be clarified. We found that mouse lacking DAO activity showed accumulation of systemic IgA. Analysis of intestinal microbiota in DAO null mice revealed increased amount of specific bacteria that stimulated IgA production. Crossbreeding experiment between DAO null mice and T cell receptor double knockout (Tcrb−/−, Tcrd−/−) mice showed that the hyper production of IgA is caused by T cell dependent B cell activation. IgA nephropathy (IgAN) occasionally causes chronic kidney impairment. Recent genome wide association analysis indicated the linkage between IgAN and genes associated with mucosal immunity. We found that high IgA (HIGA) mouse, an animal model of IgAN, lacked DAO activity, and that high plasma IgA in HIGA mice is dependent on intestinal microbiota. These results indicated that loss of DAO activity involves in pathogenesis of IgAN through disturbance of mucosal immunity.

Variations of L- and D-amino acid levels in the brain of wild-type and mutant mice lacking D-amino acid oxidase activity.

D-amino acids are now recognized to be widely present in organisms and play essential roles in biological processes. Some D-amino acids are metabolized by D-amino acid oxidase (DAO), while D-Asp and D-Glu are metabolized by D-aspartate oxidase (DDO). In this study, levels of 22 amino acids and the enantiomeric compositions of the 19 chiral proteogenic entities have been determined in the whole brain of wild-type ddY mice (ddY/DAO+/+), mutant mice lacking DAO activity (ddY/DAO-/-), and the heterozygous mice (ddY/DAO+/-) using high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). No significant differences were observed for L-amino acid levels among the three strains except for L-Trp which was markedly elevated in the DAO+/- and DAO-/- mice. The question arises as to whether this is an unknown effect of DAO inactivity. The three highest levels of L-amino acids were L-Glu, L-Asp, and L-Gln in all the three strains. The lowest L-amino acid level was L-Cys in ddY/DAO+/- and ddY/DAO-/- mice, while L-Trp showed the lowest level in ddY/DAO+/+mice. The highest concentration of D-amino acid was found to be D-Ser, which also had the highest % D value (~ 25%). D-Glu had the lowest % D value (~ 0.01%) in all the three strains. Significant differences of D-Leu, D-Ala, D-Ser, D-Arg, and D-Ile were observed in ddY/DAO+/- and ddY/DAO-/- mice compared to ddY/DAO+/+ mice. This work provides the most complete baseline analysis of L- and D-amino acids in the brains of ddY/DAO+/+, ddY/DAO+/-, and ddY/DAO-/- mice yet reported. It also provides the most effective and efficient analytical approach for measuring these analytes in biological samples. This study provides fundamental information on the role of DAO in the brain and may be relevant for future development involving novel drugs for DAO regulation.

Mouse d-Amino-Acid Oxidase: Distribution and Physiological Substrates.

d-Amino-acid oxidase (DAO) catalyzes the oxidative deamination of d-amino acids. DAO is present in a wide variety of organisms and has important roles. Here, we review the distribution and physiological substrates of mouse DAO. Mouse DAO is present in the kidney, brain, and spinal cord, like DAOs in other mammals. However, in contrast to other animals, it is not present in the mouse liver. Recently, DAO has been detected in the neutrophils, retina, and small intestine in mice. To determine the physiological substrates of mouse DAO, mutant mice lacking DAO activity are helpful. As DAO has wide substrate specificity and degrades various d-amino acids, many d-amino acids accumulate in the tissues and body fluids of the mutant mice. These amino acids are d-methionine, d-alanine, d-serine, d-leucine, d-proline, d-phenylalanine, d-tyrosine, and d-citrulline. Even in wild-type mice, administration of DAO inhibitors elevates D-serine levels in the plasma and brain. Among the above d-amino acids, the main physiological substrates of mouse DAO are d-alanine and d-serine. These two d-amino acids are most abundant in the tissues and body fluids of mice. d-Alanine derives from bacteria and produces bactericidal reactive oxygen species by the action of DAO. d-Serine is synthesized by serine racemase and is present especially in the central nervous system, where it serves as a neuromodulator. DAO is responsible for the metabolism of d-serine. Since DAO has been implicated in the etiology of neuropsychiatric diseases, mouse DAO has been used as a representative model. Recent reports, however, suggest that mouse DAO is different from human DAO with respect to important properties.

Two-dimensional high-performance liquid chromatographic determination of day–night variation of d-alanine in mammals and factors controlling the circadian changes

D-Alanine (D-Ala) is one of the naturally occurring D-amino acids in mammals, and its amount is known to have characteristic circadian changes. It is a candidate for a novel physiologically active substance and/or a biomarker, and the regulation mechanisms of the intrinsic amounts of D-Ala are expected to be clarified. In the present study, the effects of the possible factors controlling the D-Ala amounts, e.g., diet, D-amino acid oxidase (DAO) and intestinal bacteria, on the day-night changes in the intrinsic D-Ala amounts have been investigated using a highly sensitive and selective two-dimensional high-performance liquid chromatographic system combining a reversed-phase column and an enantioselective column. The circadian rhythm was not changed under fasting conditions. In the mice lacking D-amino acid oxidase activity (ddY/DAO(-) mice), clear day-night changes were still observed, suggesting that the factors controlling the D-Ala rhythm were not their food and DAO activity. On the other hand, in the germ-free mice, quite low amounts of D-Ala were detected compared with those in the control mice, indicating that the main origin of D-Ala in the mice is intestinal bacteria. Because the D-Ala amounts in the digesta containing intestinal bacteria did not show the day-night changes, the controlling factor of the circadian changes of the D-Ala amount was suggested to be the intestinal absorption.

D-Amino acids in the brain and mutant rodents lacking d-amino-acid oxidase activity

D-Amino acids are stereoisomers of L-amino acids. They are often called unnatural amino acids, but several D-amino acids have been found in mammalian brains. Among them, D-serine is abundant in the forebrain and functions as a co-agonist of NMDA receptors to enhance neurotransmission. D-Amino-acid oxidase (DAO), which degrades neutral and basic D-amino acids, is mainly present in the hindbrain. DAO catabolizes D-serine and, therefore, modulates neurotransmission. In the brains of mutant mice and rats lacking DAO activity, the amounts of D-serine and other D-amino acids are markedly increased. Mutant mice manifested behavioral changes characteristic of altered NMDA receptor activity, likely due to increased levels of D-serine. D-Serine and DAO have been demonstrated to play important roles in cerebellar development and synaptic plasticity. They have also implicated in amyotrophic lateral sclerosis and pain response. There have also been several lines of evidence correlating DAO with schizophrenia. Taken together, the experiments indicate that D-amino acids and DAO have pivotal functions in the central nervous system.

Behavioral characterization of a mutant mouse strain lacking d-amino acid oxidase activity

d-amino acid oxidase (DAO), an enzyme that degrades d-serine, has been suggested as a susceptibility factor for schizophrenia. Here we sought to understand more about the behavioral consequence of lacking DAO and the potential therapeutic implication of DAO inhibition by characterizing a mouse strain (ddY/DAO −) lacking DAO activity. We found that the mutant mice showed enhanced prepulse inhibition responses (PPI). Intriguingly, DAO−/− mice had increased sensitivity to the PPI-disruptive effect induced by the competitive NMDA antagonist, SDZ 220-581. In the 24-h inhibitory avoidance test, DAO−/− mice were not different from DAO+/+ mice during the recall. In Barnes Maze, we found that DAO−/− mice had a shortened latency to enter the escape tunnel. Interestingly, although these mice were hypoactive when tested in a protected open field, they showed a profound increase of activity on the edge of the unprotected open field of the Barnes Maze even with the escape tunnel removed. This increased edge activity does not appear to be related to a reduced level of anxiety given that there were no significant genotype effects on the measures of anxiety-like behaviors in two standard animal models of anxiety, elevated plus maze and novelty suppressed feeding. Our data suggest that DAO−/− mice might have altered functioning of NMDARs. However, these results provide only modest support for manipulations of DAO activity as a potential therapeutic approach to treat schizophrenia.

Mice lacking d-amino acid oxidase activity exhibit marked reduction of methamphetamine-induced stereotypy

The behavioral effects induced by methamphetamine (5.0 mg/kg) were compared in the mutant mice lacking d-amino acid oxidase activity and normal mice. The mutant mice exhibited marked decline in the methamphetamine-induced stereotypy compared to the normal mice, whereas the mutant mice displayed a drastic augmentation in the locomotor activity evoked by methamphetamine compared to the normal mice. Because the d-serine levels in the brain of the mutant mice are significantly higher than those in the normal mice, the enhanced d-serine in the brain of the mutant mice could antagonize the methamphetamine-induced stereotypy via the N-methyl- d-aspartate receptors.

Presence and origin of large amounts of d-proline in the urine of mutant mice lacking d-amino acid oxidase activity

Using a column-switching HPLC system combining a micro-ODS column and a chiral column, the amounts of D-proline (D-Pro) have been determined in 18 tissues, plasma and urine of mice. To avoid the enzymatic degradation of D-amino acids in vivo, a mutant mouse strain lacking D-amino acid oxidase activity (ddY/DAO(-) mouse) was used. In the brain, relatively large amounts of D-Pro were observed in the anterior pituitary, posterior pituitary and pineal glands. In the peripheral tissues, the amounts of D-Pro were high in the pancreas and kidney. Above all, it is surprising that the ddY/DAO(-) mice excreted large amounts of D-Pro in their urine (433 nmol/mL, 20 times that of L-Pro). The origin of D-Pro has also been investigated. By comparing germ-free mice and gnotobiotic mice, intestinal bacteria were shown to have no effect on the urinary D-Pro amount. Concerning the dietary origin, a notable amount of D-Pro was still excreted in the urine after starvation for 4 days, suggesting that some of the D-Pro is produced in the mice. Age-dependent changes in the urinary D-Pro amount have also been investigated from the postnatal 1st month up to 12 months, and ddY/DAO(-) mice were found to excrete large amounts of D-Pro in the urine constantly throughout their lives.

Behavioral and biochemical characterization of a mutant mouse strain lacking d-amino acid oxidase activity and its implications for schizophrenia

d-Amino acid oxidase (DAO) degrades d-serine, a co-agonist at the NMDA receptor (NMDAR). Hypofunction of the NMDAR has been suggested to contribute to the pathophysiology of schizophrenia. Intriguingly, DAO has been recently identified as a risk factor for schizophrenia through genetic association studies. A naturally occurring mouse strain (ddY/DAO −) has been identified which lacks DAO activity. We have characterized this strain both behaviorally and biochemically to evaluate DAO as a target for schizophrenia. We have confirmed that this strain lacks DAO activity and shown for the first time it has increased occupancy of the NMDAR glycine site due to elevated extracellular d-serine levels and has enhanced NMDAR function in vivo. Furthermore, the ddY/DAO − strain displays behaviors which suggest that it will be a useful tool for evaluation of the clinical benefit of DAO inhibition in schizophrenia.

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.

Determination of Free [formula omitted]-Proline and [formula omitted]-Leucine in the Brains of Mutant Mice Lacking [formula omitted]-Amino Acid Oxidase Activity

A new procedure to accurately measure a trace amount of d-proline in biological samples has been developed. This d-amino acid was derivatized with 4-fluoro-7-nitro-2,1,3-benzoxadiazole and was determined by a column-switching HPLC system, a combination of a micro-ODS column and a chiral column. The detection limit for d-proline spiked in a mouse cerebrum sample is 1 fmol (injection amount, S/N = 3). Within-day precision and day-to-day precision obtained for spiked d-proline (10 fmol) are 2.14 and 5.35% (RSD), respectively. Using the new method, the amount of free d-proline in eight brain regions and sera of mutant ddY/DAO− mice, lacking d-amino acid oxidase activity, and control ddY/DAO+ mice was determined. The amount of free d-leucine was also investigated. The amount and distribution of d-proline in the brains of ddY/DAO+ mice and ddY/DAO− mice are almost the same, and relatively high amounts of d-proline have been observed in the pituitary gland and in the pineal gland. On the other hand, the amount of d-leucine is different between the two strains. In the brains of ddY/DAO+ mice, a relatively high amount of d-leucine has been observed in the pineal gland compared with other regions. In the brains of ddY/DAO− mice, d-leucine amounts are approximately 10 times higher than those obtained in ddY/DAO+ mice and regional difference has not been observed, while the amounts of l-proline and l-leucine are not significantly different between the two strains. In the serum, the amounts of both free d-proline and d-leucine are significantly higher in the ddY/DAO− mice than those obtained in ddY/DAO+ mice.

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.

Involvement of D-amino acid oxidase in elimination of D-serine in mouse brain.

The physiological role of D-amino acid oxidase (EC 1. 4. 3. 3) in mouse brain is described. The presence of D-enantiomers of neutral common amino acids was surveyed in the brain. D-serine was shown to be present at high concentration only in regions where the enzyme activity was low. In normal mice whose D-amino acid oxidase activity was much higher in the cerebellum than in the cerebrum, free D-serine content was apparently lower in the cerebellum than in the cerebrum. In mice of a mutant strain lacking D-amino acid-oxidase activity, the free D-serine level was remarkably high both in the cerebrum and cerebellum. The results suggest that the enzyme is involved in the elimination of free D-serine in the cerebellum.