The gliotransmitter D-serine is released upon (S)-␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid͞kainate and metabotropic glutamate receptor stimulation, but the mechanisms involved are unknown. Here, by using a highly sensitive bioassay to continuously monitor extracellular D-serine levels, we have investigated the pathways used in its release. We reveal that D-serine release is inhibited by removal of extracellular calcium and augmented by increasing extracellular calcium or after treatment with the Ca 2؉ ionophore A23187. Furthermore, release of the amino acid is considerably reduced after depletion of thapsigargin-sensitive intracellular Ca 2؉ stores or chelation of intracellular Ca 2؉ with 1,2-bis(2-aminophenoxy)ethane-N,N,N ,N -tetraacetate-acetoxymethyl ester. Interestingly, D-serine release also was markedly reduced by concanamycin A, a vacuolar-type H ؉ -ATPase inhibitor, indicating a role for the vesicular proton gradient in the transmitter storage͞release. In addition, agonist-evoked D-serine release was sensitive to tetanus neurotoxin. Finally, immunocytochemical and sucrose density gradient analysis revealed that a large fraction of D-serine colocalized with synaptobrevin͞VAMP2, suggesting that it is stored in VAMP2-bearing vesicles. In summary, our study reveals the cellular mechanisms subserving D-serine release and highlights the importance of the glial cell exocytotic pathway in influencing CNS levels of extracellular D-serine.glia ͉ synaptobrevin ͉ D-amino acid ͉ vesicles ͉ tetanus neurotoxin A strocytes play pivotal roles in synaptic transmission by controlling transmitter diffusion and concentration in the extracellular space (1) and also by back-signaling to neurons directly through the release of neuroactive substances (2). Although glutamate and ATP are the most recognized chemical transmitters that mediate astrocyte-neuron signaling (2), other cell-cell mediators also are involved in this pathway. D-Serine has recently been identified as a major gliotransmitter in the central nervous system that serves as an endogenous ligand for the glycine site of NMDA receptors (3-6).However, many aspects regarding D-serine release still need to be addressed. Although it has been suggested that astrocytes may release D-serine, through the reverse operation of a sodiumdependent transporter (7), the precise molecular mechanisms underlying D-serine release are currently unknown. To unravel the functional consequences of D-serine-mediated astrocyte-toneuron 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 bioassay to continuously monitor the release of D-serine from cultured glial cells. In this work, we show that astrocytes and C6 glioma cells synthesize and contain a large amount of D-serine that can be released upon glutamate receptor (GluR) stimulation. Moreover, our observations demonstrate that D-serine release, evoked by glutamatergic agonists, is linked to a [Ca 2ϩ...

Human genes coding for pLG72 and D-amino acid oxidase have recently been linked to the onset of schizophrenia. pLG72 was proposed as an activator of the human FAD-containing flavoprotein D-amino acid oxidase (hDAAO). In the brain this oxidizes D-serine, a potent activator of N-methyl-D-aspartate receptor. We have investigated the mechanistic regulation of hDAAO by pLG72. Immunohistochemical analyses revealed that hDAAO and pLG72 are both expressed in astrocytes of the human cortex, where they most likely interact, considering their partial overlapping subcellular distribution and their coimmunoprecipitation. We demonstrated that the specific in vitro interaction of the two proteins yields a complex composed of 2 hDAAO homodimers and 2 pLG72 molecules. Binding of pLG72 did not affect the kinetic properties and FAD binding ability of hDAAO; instead, a time-dependent loss of hDAAO activity in the presence of an excess of pLG72 was found. The binding affects the tertiary structure of hDAAO, altering the amount of the active form. We finally demonstrated that overexpression of hDAAO in glioblastoma cells decreases the levels of D-serine, an effect that is null when pLG72 is coexpressed. These data indicate that pLG72 acts as a negative effector of hDAAO. Therefore, a decrease in the synaptic concentration of D-serine as the result of an anomalous increase in hDAAO activity related to hypoexpression of pLG72 may represent a molecular mechanism by which hDAAO and pLG72 are involved in schizophrenia susceptibility.Schizophrenia is one of the most widely spread psychiatric disorders; it is a complex disease or, more likely, a group of related illnesses to which an individual has a strong genetic predisposition (1). Among the identified schizophrenia susceptibility genes (2), the gene G72 encodes for several splicing isoforms; pLG72 represents the longest open reading frame (153 amino acids), which is mainly expressed in brain (3). G72 is present only in primates: there are no homologues of this gene in databases nor has sequence analysis of the putative open reading frame revealed any likely function (2, 3). Yeast twohybrid experiments using pLG72 as bait identified D-amino acid oxidase (EC 1.4.3.3, DAAO 4 ) on 12q24 as a putative interacting partner, and preliminary functional measurements showed that pLG72 should function as an in vitro activator of pig kidney DAAO (pkDAAO) (3). DAAO is a FAD-containing flavoenzyme that catalyzes the oxidative deamination of D-amino acids to the corresponding ␣-keto acids, hydrogen peroxide and ammonia (4, 5).Based on current findings we can hypothesize that in brain, the physiological role of DAAO is to modulate the levels of D-serine, an important glial-derived messenger that acts as the endogenous allosteric modulator of the glutamatergic NMDA receptor subtype (6 -8). D-and L-serine can be reversibly isomerized in astrocytic glia, which unsheathes synapses, by serine racemase. Compelling evidence has indicated that glutamate neurotransmission hypofunction is associated with symptoms...

Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signalling pathways. In particular, astrocytes have recently been shown to release small molecules such as the amino acids l-glutamate and d-serine as “gliotransmitters”, which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca2+-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles including the SNARE synaptobrevin 2 and contain both l-glutamate and d-serine. Furthermore, they show uptake of l-glutamate and d-serine that is driven by a proton electrochemical gradient. d-Serine uptake is associated with vesicle acidification and is dependent on chloride. While l-serine is not transported, serine racemase, the synthesizing enzyme for d-serine, is anchored to the membrane of the vesicles allowing local generation of d-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between d-serine and l-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing d-serine, l-glutamate, and most likely other neuromodulators in an activity-dependent manner.