D-Amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidation of d-amino acids. In the brain, gene expression of DAO is detected in astrocytes. Among the possible substrates of DAO in vivo, d-serine is proposed to be a neuromodulator of the N-methyl-d-aspartate (NMDA) receptor. In a search for the physiological role of DAO in the brain, we investigated the metabolism of extracellular d-serine in glial cells. Here we show that after d-serine treatment, rat primary type-1 astrocytes exhibited increased cell death. In order to enhance the enzyme activity of DAO in cells, we established stable rat C6 glial cells overexpressing mouse DAO designated as C6/DAO. Treatment with a high dose of d-serine led to the production of hydrogen peroxide (H₂O₂) followed by apoptosis in C6/DAO cells. Among the amino acids tested, d-serine specifically exhibited a significant cell death–inducing effect. DAO inhibitors, i.e., sodium benzoate and chlorpromazine, partially prevented the death of C6/DAO cells treated with d-serine, indicating the involvement of DAO activity in d-serine metabolism. Overall, we consider that extracellular d-serine can gain access to intracellular DAO, being metabolized to produce H₂O₂. These results support the proposal that astroglial DAO plays an important role in metabolizing a neuromodulator, d-serine.

D-Amino acid oxidase (DAO), a potential risk factor for schizophrenia, has been proposed to be involved in the decreased glutamatergic neurotransmission in schizophrenia. Here we show the inhibitory effect of an antipsychotic drug, chlorpromazine, on human DAO, which is consistent with previous reports using porcine DAO, although human DAO was inhibited to a lesser degree (K_i = 0.7 mM) than porcine DAO. Since chlorpromazine is known to induce phototoxic or photoallergic reactions and also to be transformed into various metabolites, we examined the effects of white light-irradiated chlorpromazine on the enzymatic activity. Analytical methods including high-resolution mass spectrometry revealed that irradiation triggered the oligomerization of chlorpromazine molecules. The oligomerized chlorpromazine showed a mixed type inhibition with inhibition constants of low micromolar range, indicative of enhanced inhibition. Taken together, these results suggest that oligomerized chlorpromazine could act as an active substance that might contribute to the therapeutic effects of this drug.

D-Amino acid oxidase (DAO) is a flavoenzyme that exists in the kidney, liver and brain of mammals. This enzyme catalyzes the oxidation of d-amino acids to the corresponding α-keto acid, hydrogen peroxide and ammonia. Recently d-serine, one of the substrates of DAO, has been found in the mammalian brain, and shown to be a co-agonist of the N-methyl-d-aspartate (NMDA) receptor in glutamate neurotransmission. In this study, we investigated the metabolism of extracellular d-serine and the effects of d-serine metabolites to study the pathophysiological role of DAO. Treatment with a high dose of d-serine induced the cell death in dose-dependent manner in DAO-expressing cells. Moreover, overexpression of DAO in astroglial cells induced the enhanced cytotoxicity. The treatment with 1 mM beta-hydroxypyruvate (HPA), uniquely produced from the d-serine metabolism by DAO activity, also induced cell death, comprising apoptosis, in the astroglial cell, but not in the other cells derived from liver and kidney. Taken together, we consider that high dose of extracellular d-serine induced cell death by the production of not only hydrogen peroxide but also HPA as a result of DAO catalytic activity in astroglial cell. Furthermore, this cytotoxicity of HPA is observed uniquely in astroglial cells expressing DAO.

Nuclear receptor subfamily 5 group A member 1 (NR5A1) is expressed in the pituitary gonadotrope and regulates their differentiation. Although several regulatory regions were implicated in Nr5a1 gene expression in the pituitary gland, none of these regions have been verified using mouse models. Furthermore, the molecular functions of NR5A1 in the pituitary gonadotrope have not been fully elucidated. In the present study, we generated mice lacking the pituitary enhancer located in the 6th intron of the Nr5a1 gene. These mice showed pituitary gland-specific disappearance of NR5A1, confirming the functional importance of the enhancer. Enhancer-deleted male mice demonstrated no defects at fetal stages. Meanwhile, androgen production decreased markedly in adult, and postnatal development of reproductive organs, such as the seminal vesicle, prostate, and penis was severely impaired. We further performed transcriptomic analyses of the whole pituitary gland of the enhancer-deleted mice and controls, as well as gonadotropes isolated from Ad4BP-BAC-EGFP mice. These analyses identified several genes showing gonadotrope-specific, NR5A1-dependent expressions, such as Spp1, Tgfbr3l, Grem1, and Nr0b2. These factors are thought to function downstream of NR5A1 and play important roles in reproductive organ development through regulation of pituitary gonadotrope functions.