Abstract:
Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signaling pathways. In particular, astrocytes have been shown recently 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 Ca(2+)-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 (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) 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. Whereas 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.

Abstract:
D-Serine is an astrocyte-derived regulator for N-methyl-D-aspartate receptors, but the intracellular routes of its trafficking are still largely unknown. Here, we combined confocal microscopy with colocalization quantification to track the astrocytic organelles that store D-serine. We report that D-serine colocalizes with the transfected eGFP-synaptobrevin/VAMP2 and eGFP-cellubrevin/VAMP3, two v-SNAREs of the regulated secretory pathway. No significant colocalization was found with markers of the endosomal sorting and recycling system: EEA1, eGFP-endobrevin/VAMP8, eGFP-TI-VAMP/VAMP7, LAMP1, and CD63. Blockade of vesicular budding with colchicine shows that secretory vesicles import D-serine downstream to the Golgi apparatus. Finally, treatment of astrocytes with the Ca2+-ionophore A23187, glutamate agonists, or bradykinin trigger translocation of synaptobrevin/VAMP2 to the plasma membrane with a concomitant disappearance of D-serine from the regulated secretory pathway. Our results provide morphological evidence for a vesicular storage of D-serine in the regulated secretory pathway and the possible recruitment of these stores by Ca2+ mobilization to release D-serine.

Abstract:
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.

Abstract:
The patterns of development of the vestibular nuclei (VN) and their main connections involving glutamate neurotransmission offer a good model for studying the function of the glial-derived neuromodulator D-serine in synaptic plasticity. In this study we show that D-serine is present in the VN and we analyzed its distribution and the levels of expression of serine racemase and D-amino acid oxidase (D-AAO) at different stages of postnatal (P) development. From birth to P21, high levels of D-serine were detected in glial cells and processes in all parts of the VN. This period corresponded to high expression of serine racemase and low expression of D-AAO. On the other hand, in the mature VN D-serine displayed very low levels and was mainly localized in neuronal cell bodies and dendrites. This drop of D-serine in adult stages corresponded to an increasing expression of D-AAO at mature stages. High levels of glial D-serine during the first 3 weeks of postnatal development correspond to an intense period of plasticity and synaptogenesis and maturation of VN afferents, suggesting that D-serine could be involved in these phenomena. These results demonstrate for the first time that changes in D-serine levels and distribution occur during postnatal development in the central nervous system. The strong decrease of D-serine levels and the glial-to-neuronal switch suggests that D-serine may have distinct functional roles depending on the developmental stage of the vestibular network.

Abstract:
Neurons and glia talk to each other at synapses. Glia sense the level of synaptic activity and consequently regulate its efficacy via the release of neuromodulators. One such glia-derived modulator is D-serine, an amino acid that serves as an endogenous ligand for the strychnine-insensitive glycine-binding site of NMDA glutamate receptors. Here, we provide an overview of recent findings on the mechanisms of its synthesis, release and clearance at synapses, with an emphasis on the dichotomy of behaviour of this novel messenger in the brain. The discovery of the good and ugly faces of this gliotransmitter is an important issue of modern neuroscience that has repercussions for the treatment of brain disorders.

Abstract:
Long ignored and only considered as housekeeping cells for neurons, astroglial cells in the last decade have gained increasing attention as key players of higher functions in healthy brain, but also in diseases. This revolution in our way to think the active brain culminates in the concept of a tripartite synapse, which considers glial cells and notably astrocytes as an integral dynamic partner of synapses. Glia not only listens but also talks to neurons through the release of neuroactive substances. Recently much attention has been paid to the role played by the atypical amino acid D-serine in this signalling pathway. This molecule synthesized through racemization of L-serine fulfils most criteria as a gliotransmitter and as the endogenous ligand for the strychnine-insensitive glycine binding site of the NMDA receptors. D-serine is considered to be a permissive factor for long-term changes in synaptic plasticity and neuronal migration through activation of NMDA receptors. It is also known that disturbance of NMDA receptors activity can cause cell death. Not surprisingly, then, D-serine has also been found to promote neurons death in experimental models of beta-amyloid peptide-induced neuroinflammation and of ischaemia by overactivating the NMDA receptors. Finally, in a more recent past, studies have pointed to the molecular mechanisms leading to D-serine release into and removal from the synaptic cleft.

Abstract:
The gliotransmitter D-serine is released upon (S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate 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 storage/release. 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 synaptobrevin/VAMP2, 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.