Molecular Enzymological Studies on the Pathophysiological Significance of Brain Endogeneous D-amino Acids and Their Metabolizing Enzyme

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D-amino acid oxidase (DAO) is a flavoenzyme that catalyzes the oxidative deamination of D-amino acids. Histochemical studies revealed that DAO activity was located to some types of astrocytes. Although the existence of in vivo substrate of DAO was not known for many years, recently it had been demonstrated that substantial amounts of free D-serine are present in mammalian brain. D-serine as well as giycine acts as co-agonists at glycine site of N-methyi-D-aspartate(NMDA) type of glutamate receptor which plays an important role in nurotoxicity in cerebral ischemia. Extracelluiar free D-serine can modulate neurotransmission. D-serine was found to be localized to type-2 astrocytes in culture, and released by glutamate stimulation.We examined the DAO gene expression in cultured rat astrocytes to investigate the physiological role of DAO in the context of free D-serine metabolism. We examined the DAO gene expression in cultured rat astrocytes by reverse transcriptase-polymerase chain reaction. We established a method to prepare highly purified cuiture of type-1 and type-2 astrocytes from any brain region. This method utilizes combinaion of cell type spesific separaton by shaking and subsequent purification by immunopanning or trearment with cytosine arabinoside. We detected higher DAO gene expression in type-1 astrocyte cultures from cerebellum than that in type-2, We also revealed that DAO expression in C6, corresponding to type-1astrocyte, was higher than that in CG-4 derived type-2 astrocytes. Gene expression of DAO in microglia was also demonstrated with the use of MG5 cell line and primary culture.