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Glutamate Receptor Activation Triggers a Calcium- Dependent and SNARE Protein-Dependent Release of the Gliotransmitter D-Serine

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Glutamate Receptor Activation Triggers a Calcium- Dependent and SNARE Protein-Dependent Release of the Gliotransmitter D-Serine Glutamate receptor activation triggers a calcium- dependent and SNARE protein-dependent release of the gliotransmitter D-serine Jean-Pierre Mothet*†, Loredano Pollegioni‡, Gilles Ouanounou*, Magalie Martineau*, Philippe Fossier*, and Ge´ rard Baux* *Laboratoire de Neurobiologie Cellulaire et Mole´ culaire, Centre National de la Recherche Scientifique Unite´ Propre de Recherche 9040, Institut Fe´de´ ratif de Neurobiologie Alfred Fessard, F-91198 Gif-sur-Yvette, France; and ‡Department of Biotechnology and Molecular Sciences, University of Insubria, 21100 Varese, Italy Edited by Solomon H. Snyder, Johns Hopkins University School of Medicine, Baltimore, MD, and approved February 25, 2005 (received for review November 19, 2004) The gliotransmitter D-serine is released upon (S)-␣-amino-3-hydroxy- increase, because efflux of the transmitter is altered by manip- 5-methyl-4-isoxazolepropionic acid͞kainate and metabotropic glu- ulations of intracellular and extracellular calcium ion concen- tamate receptor stimulation, but the mechanisms involved are trations. Furthermore, treatment of primary astrocytes or glioma unknown. Here, by using a highly sensitive bioassay to continu- cells with tetanus neurotoxin (TeNT), which selectively cleaves ously monitor extracellular D-serine levels, we have investigated the vesicle-associated SNARE proteins VAMP2 and VAMP3 the pathways used in its release. We reveal that D-serine release is (8, 9), or with concanamycin A, a compound that inhibits the inhibited by removal of extracellular calcium and augmented by vacuolar-type Hϩ-ATPase (10), strongly reduced the release of ؉ increasing extracellular calcium or after treatment with the Ca2 D-serine. Finally, we demonstrate that a large part of D-serine is ionophore A23187. Furthermore, release of the amino acid is stored in VAMP2-bearing vesicles, as revealed by sucrose- considerably reduced after depletion of thapsigargin-sensitive gradient analysis and confocal microscopy. We thus provide, to intracellular Ca2؉ stores or chelation of intracellular Ca2؉ with our knowledge, the first evidence for vesicular storage and ϩ .1,2-bis(2-aminophenoxy)ethane-N,N,N؅,N؅-tetraacetate–acetoxy- Ca2 -dependent exocytotic release of D-serine from glial cells methyl ester. Interestingly, D-serine release also was markedly reduced by concanamycin A, a vacuolar-type H؉-ATPase inhibitor, Materials and Methods indicating a role for the vesicular proton gradient in the transmitter Cell Culture. Cultured astrocytes were prepared from cerebral storage͞release. In addition, agonist-evoked D-serine release was cortex of 1- to 3-day-old postnatal rats, as described in ref. 11, sensitive to tetanus neurotoxin. Finally, immunocytochemical and and used after 1–3 weeks in culture. Rat glioma C6 cells were sucrose density gradient analysis revealed that a large fraction of purchased from American Type Culture Collection and cultured D-serine colocalized with synaptobrevin͞VAMP2, suggesting that it in DMEM͞Nut mix F-12 (Invitrogen) supplemented with 2 mM is stored in VAMP2-bearing vesicles. In summary, our study reveals glutamine and 10% FCS. Cells were grown at 37°C in a 5% CO2 the cellular mechanisms subserving D-serine release and highlights incubator. the importance of the glial cell exocytotic pathway in influencing CNS levels of extracellular D-serine. Bioassay for D-Serine Release. D-Serine levels were detected by using an enzymatic assay (12) (Fig. 1A). After the addition of glia ͉ synaptobrevin ͉ D-amino acid ͉ vesicles ͉ tetanus neurotoxin D-amino acid oxidase (DAAO) and horseradish peroxidase (HRP)͞luminol to astrocytes bathed in saline solution, any strocytes play pivotal roles in synaptic transmission by detectable levels of D-serine released in the medium were Acontrolling transmitter diffusion and concentration in the visualized by an emission of light. For this bioassay, DAAO extracellular space (1) and also by back-signaling to neurons purified from the yeast Rhodotorula gracilis (RgDAAO) was directly through the release of neuroactive substances (2). used. RgDAAO displays peculiar features in comparison with Although glutamate and ATP are the most recognized chemical mammalian enzyme, such as the tight binding of the coenzyme ϭ ϫ Ϫ8 transmitters that mediate astrocyte–neuron signaling (2), other FAD (Kd 2.0 10 M), a strict stereospecificity for d-amino cell–cell mediators also are involved in this pathway. D-Serine acids, and a significant higher catalytic efficiency for its sub- has recently been identified as a major gliotransmitter in the strates (13). Recombinant wild-type and mutant Arg-285 3 Ala central nervous system that serves as an endogenous ligand for RgDAAOs were expressed and purified from Escherichia coli the glycine site of NMDA receptors (3–6). cells by using the pT7-DAAO expression system in However, many aspects regarding D-serine release still need to BL21(DE3)pLysS cells (14). The mutant enzyme shows a resid- be addressed. Although it has been suggested that astrocytes may ual activity of Ͻ0.1% of that determined for the wild-type 3 release D-serine, through the reverse operation of a sodium- DAAO (general properties of Arg-285 Ala RgDAAO are dependent transporter (7), the precise molecular mechanisms described in ref. 15). The presence of the HRP and luminol does underlying D-serine release are currently unknown. To unravel not affect the activity of the RgDAAO (see Fig. 7, which is the functional consequences of D-serine-mediated astrocyte-to- published as supporting information on the PNAS web site). neuron signaling, it is essential to shed light on the mechanisms controlling the gliotransmitter storage and release pathways. For this purpose, we have devised a previously undescribed This paper was submitted directly (Track II) to the PNAS office. ␣ bioassay to continuously monitor the release of D-serine from Abbreviations: AMPA, (S)- -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; CgB, chromogranin B; DAAO, d-amino acid oxidase; HRP, horseradish peroxidase; KAR, kainate cultured glial cells. In this work, we show that astrocytes and C6 receptor; LDCL, luminol-derived chemiluminescence; GluR, glutamate receptor; mGluR, glioma cells synthesize and contain a large amount of D-serine metabotropic GluR; RgDAAO, DAAO purified from Rhodotorula gracilis; t-ACPD, (1S,3R)- that can be released upon glutamate receptor (GluR) stimula- 1-aminocyclopentane-1,3-dicarboxylic acid. tion. Moreover, our observations demonstrate that D-serine †To whom correspondence should be addressed. E-mail: [email protected]. 2ϩ release, evoked by glutamatergic agonists, is linked to a [Ca ]i © 2005 by The National Academy of Sciences of the USA 5606–5611 ͉ PNAS ͉ April 12, 2005 ͉ vol. 102 ͉ no. 15 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0408483102 ϩ NAD assay (16), and D-serine content was measured by using the chemiluminescent assay in offline mode. Protein Electrophoresis and Immunoblotting. Protein extracts resus- pended in denaturating sample buffer were subjected to SDS͞ PAGE (12%) analysis, followed by blotting onto poly(vinylidene difluoride) membrane. Immunodetection was performed by using the ECL amplification system (Amersham Pharmacia Biotech) following the manufacturer’s protocol. Protein content was deter- mined by the Lowry method using the DC protein Bio-Rad assay. Immunostaining. Subconfluent cell cultures prepared as described above were fixed in 4% paraformaldehyde͞0.1% glutaraldehyde for 60 min before being treated with blocking solution containing 4% horse serum and 0.2% Triton X-100 for1hatroom temperature. Cultures then were probed with anti-glial fibrillary acidic protein (1:2,000, DAKO), conjugated anti-D-serine (1:2,000, GEMAC, Cenon, France), anti-VAMP2 (1:1,000, Synaptic System, Gottin- gen, Germany), anti-VAMP3 antibody (1:100, Santa Cruz Biotech- nology), or anti-chromogranin B (CgB) (1:100, courtesy of J. Meldolesi, San Raffaele Scientific Institute, Milan) overnight at 4°C. After washing to remove excess primary antibodies, the cultures were incubated for1hatroom temperature with Alexa secondary antibodies (Molecular Probes). Cells were imaged by using an upright laser-scanning confocal microscope (TCS SP2, Leica, Mannheim, Germany). Controls were performed by antisera preadsorption with 0.5 mM liquid D-serine-glutaraldehyde conju- gate or by emitting the primary antibody. Colocalization of D-serine Fig. 1. Schematic illustration and standard curves of the D-serine-induced with different cellular markers was quantified with IMAGEJ software reaction pathway leading to luminol-derived chemiluminescence (LDCL) with ͞͞ ͞ the RgDAAO͞HRP enzymatic system. (A) Recombinant RgDAAO catalyzes the (http: rsb.info.nih.gov ij) by using the colocalization option. Co- oxidative deamination of D-serine, producing ammonia, hydroxypyruvate, localization analysis was performed on each z-optical section for a and hydrogen peroxide (H2O2). The latter product is used by HRP to oxidize cell, and values from 10–15 different cells from three independent luminol, thus yielding a photon. The emitted photon then is captured by the experiments were used to calculate the colocalization frequencies. photomultiplier tube. (B) Examples of LDCL spectra induced by D-serine at 40 pM (smaller trace) and 50 nM (larger trace). (C) Dose-response curve for Statistics. All data were expressed as mean Ϯ SEM, and n refers integrated photons for low and high (Inset) D-serine concentrations obtained to the number of independent experiments. Statistical differ- NEUROSCIENCE with
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