Neuron-derived D-Serine Release Provides a Novel Means to Activate N-Methyl-D-aspartate Receptors

Abstract

d-Serine is a coagonist of N-methyl-d-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of d-serine is carried out by serine racemase, which converts l-to d-serine. d-Serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of d-serine. We now report significant amounts of serine racemase and d-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and d-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and d-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived d-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous d-serine by addition of the recombinant enzyme d-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal d-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked d-serine release. Release of d-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. d-Serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of d-serine. Glutamate-induced neuronal d-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way. d-Serine is a coagonist of N-methyl-d-aspartate (NMDA) receptors that occurs at high levels in the brain. Biosynthesis of d-serine is carried out by serine racemase, which converts l-to d-serine. d-Serine has been demonstrated to occur in glial cells, leading to the proposal that astrocytes are the only source of d-serine. We now report significant amounts of serine racemase and d-serine in primary neuronal cultures and neurons in vivo. Several neuronal culture types expressed serine racemase, and d-serine synthesis was comparable with that in glial cultures. Immunohistochemical staining of brain sections with new antibodies revealed the presence of serine racemase and d-serine in neurons. Cortical neurons expressing serine racemase also expressed the NR2a subunit in situ. Neuron-derived d-serine contributes to NMDA receptor activation in cortical neuronal cultures. Degradation of endogenous d-serine by addition of the recombinant enzyme d-serine deaminase diminished NMDA-elicited excitotoxicity. Release of neuronal d-serine was mediated by ionotropic glutamate receptor agonists such as NMDA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, and kainate. Removal of either external Ca2+ or Na+ blocked d-serine release. Release of d-serine was mostly through a cytosolic route because it was insensitive to bafilomycin A1, a potent inhibitor of vesicular neurotransmitter uptake. d-Serine was also not transported into purified synaptic vesicles under conditions optimal for the uptake of known transmitters. Our results suggest that neurons are a major source of d-serine. Glutamate-induced neuronal d-serine release provides a novel mechanism for activating NMDA receptors by an autocrine or paracrine way. The N-methyl-d-aspartate (NMDA) 2The abbreviations used are: NMDA, N-methyl-d-aspartate; AMPA, amino-3-hydroxy-5-methylisoxazole-4-propionic acid; GFAP, glial fibrillary acidic protein; MAP2, microtubule-associated protein-2; P, postnatal day; HBSS, HEPES-buffered salt solution; HPLC, high pressure liquid chromatography; TBS, Tris-buffered saline; HCSS, HEPES control salt solution; MOPS, 4-morpholinepropanesulfonic acid. type of glutamate receptor plays prominent roles in excitatory neurotransmission (1Danysz W. Parsons A.C. Pharmacol. Rev. 1998; 50: 597-664PubMed Google Scholar). In addition to glutamate, the NMDA receptor requires the obligatory binding of a coagonist of the NR1 subunit to mediate ion influx (2Johnson J.W. Ascher P. Nature. 1987; 325: 529-531Crossref PubMed Scopus (2701) Google Scholar). Although glycine was originally suggested as the NMDA receptor coagonist, recent data indicate that endogenous d-serine is a physiologically relevant NMDA receptor ligand at the coagonist site. d-Serine is present at high levels in the brain, with little levels in peripheral tissues (3Hashimoto A. Nishikawa T. Hayashi T. Fujii N. Harada K. Oka T. Takahashi K. FEBS Lett. 1992; 296: 33-36Crossref PubMed Scopus (410) Google Scholar, 4Hashimoto A. Nishikawa T. Oka T. Takahashi K. J. Neurochem. 1993; 60: 783-786Crossref PubMed Scopus (346) Google Scholar, 5Wolosker H. Panizzutti R. De Miranda J. Neurochem. Int. 2002; 41: 327-332Crossref PubMed Scopus (84) Google Scholar). Destruction of endogenous d-serine by d-amino-acid oxidase in hippocampal cultures promotes a decrease in NMDA receptor responses (6Mothet J.P. Parent A.T. Wolosker H. Brady Jr., R.O. Linden D.J. Ferris C.D. Rogawski M.A. Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar). NMDA responses in the and of hippocampal are also diminished by d-serine W. W. Proc. Natl. A. PubMed Scopus Google Scholar, Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar). d-serine was to for in the H. A. Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar). has been that glial release d-serine and through activation of NMDA receptors in H. A. Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar). In hippocampal endogenous d-serine was to the coagonist for NMDA by NMDA Wolosker H. J. PubMed Scopus Google Scholar). d-Serine is by serine racemase, a brain enzyme Miranda J. A. Wolosker H. PubMed Scopus Google Scholar, H. Takahashi J.P. Brady Jr., R.O. Ferris C.D. Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar, H. Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar). racemase is by and the enzyme also of serine into and De Miranda J. Panizzutti R. P. Wolosker H. J. PubMed Scopus Google Scholar, Miranda J. Panizzutti R. Wolosker H. Proc. Natl. A. 2002; PubMed Scopus Google Scholar). d-serine and serine racemase to in astrocytes H. Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar, Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar). cultures release d-serine receptor activation Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar, Panizzutti R. De Miranda J. Wolosker H. 2002; PubMed Scopus Google Scholar, J.P. P. Proc. Natl. A. PubMed Scopus Google and through an mechanism by the Panizzutti R. De Miranda J. Wolosker H. 2002; PubMed Scopus Google Scholar). has been that d-serine astrocytes that the neuronal NMDA receptors Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar). The that d-serine a receptor as as a and that d-serine is an in the brain H. Panizzutti R. De Miranda J. Neurochem. Int. 2002; 41: 327-332Crossref PubMed Scopus (84) Google Scholar, S.H. Ferris C.D. J. PubMed Scopus Google Scholar). the the glial of d-serine is not with the of which present in neurons. We a neuronal of d-serine and serine racemase in cultures and new antibodies d-serine and serine We the presence of serine racemase and d-serine in neurons. release of d-serine a significant of NMDA-elicited in cortical cultures. Our data indicate that neuronal d-serine an in NMDA receptor such as occurs in and d-serine AMPA, acid, bafilomycin A1, fibrillary acidic and a in NMDA, and and was was and inhibitor and was and was by neuronal cultures the and and hippocampal cultures postnatal as J. 1993; PubMed Scopus Google with the in of HEPES-buffered salt and and and for at with at a of inhibitor and of and the was with a Ca2+ and and to the and the in at a of the with The culture of with and d-serine synthesis the cultures used The culture not or was to the and the cultures used glial cultures by the with and the with to to neuronal and cultures used and glial cultures of the as that of of the culture was and the for as Miranda J. Panizzutti R. Wolosker H. Proc. Natl. A. 2002; PubMed Scopus Google Scholar). by addition of to a The was at for and the was by of by with the culture that to d-serine and with the d-serine deaminase to d-serine levels and in the in d-serine synthesis was with d-amino-acid oxidase to d-serine as Miranda J. Panizzutti R. Wolosker H. Proc. Natl. A. 2002; PubMed Scopus Google Scholar). and serine racemase was as a in as R. De Miranda J. Wolosker H. Proc. Natl. A. PubMed Scopus Google and used to The was purified a to of recombinant serine racemase that was expressed and purified as De Miranda J. Panizzutti R. P. Wolosker H. J. PubMed Scopus Google Scholar). The serine racemase was and in a H. Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar). carried out with the purified antibodies used at and the revealed with and with and with racemase carried out by of the with of purified and antibodies with neuronal and glial and was by of the of glial in the in in was by with for serine racemase at or with and with to the the was to in and with in sections a and to and in Tris-buffered and racemase was used at and in was used at in at the with and was with as the the sections and blocked as and the primary antibodies used and antibodies and racemase was revealed by at and serine racemase was revealed by by to The under a for d-serine the Snyder S.H. Proc. Natl. A. PubMed Scopus Google was as with at with and in and with in sections a or We used sections of of because staining and sections with and for the sections to blocked and with and in for the sections for with or the by Snyder S.H. Proc. Natl. A. 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Google Scholar). the of the with a racemase to the enzyme in postnatal glial cultures H. Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar). an that neuronal and glial serine to of of serine racemase was by In neuronal cultures the neurons for serine racemase, with with the neuronal hippocampal cultures also neuronal serine racemase staining We also purified neuronal cultures the and for revealed which present in and in the glial in the neuronal cultures little serine In glial cells, racemase staining was staining was in for serine racemase in cultures and that glial not the source for d-serine synthesis in neuronal cultures. 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In it is that Ca2+ influx is for d-serine release through a or to the for d-serine release. major the roles of the neuronal glial of d-serine. neurons expressing serine racemase also NMDA it is that neuronal d-serine an autocrine or paracrine activation of NMDA The presence of neuronal d-serine not that a Our neurons because are d-serine and serine racemase are present in not We neuronal d-serine only in the in brain glial staining was In glial d-serine a prominent because glial neurons in the of the glial of d-serine is the neuronal in the by Snyder and Snyder S.H. Proc. Natl. A. PubMed Scopus Google Scholar, Snyder S.H. Rev. PubMed Scopus Google glial d-serine in NMDA receptor by plays a in neurotransmission Rev. PubMed Scopus Google glial d-serine to in not neurotransmitter neurons and been demonstrated for the neurotransmitter glutamate Rev. 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