PLG72 Modulates Intracellular D-Serine Levels through Its Interaction with D-Amino Acid Oxidase : EFFECT ON SCHIZOPHRENIA SUSCEPTIBILITY

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

Caractérisation pharmacologique et moléculaire de la libération et du transport vésiculaire de la D-sérine dans les astrocytes

LE KREMLIN-B.- PARIS 11-BU Méd (940432101) / SudocSudocFranceF

d-Aspartate: An endogenous NMDA receptor agonist enriched in the developing brain with potential involvement in schizophrenia

Free d-aspartate and d-serine occur at substantial levels in the mammalian brain. d-Serine is a physiological endogenous co-agonist for synaptic N-Methyl d-Aspartate (NMDA) receptors (NMDARs), and is involved in the pathophysiology of schizophrenia. Much less is known about the biological meaning of d-aspartate. d-Aspartate is present at high levels in the embryo brain and strongly decreases at post-natal phases. Temporal reduction of d-aspartate levels depends on the post-natal onset of d-aspartate oxidase (DDO), an enzyme able to selectively catabolize this d-amino acid. Pharmacological evidence indicates that d-aspartate binds to and activates NMDARs. Characterization of genetic and pharmacological mouse models with abnormally higher levels of d-aspartate has evidenced that increased d-aspartate enhances hippocampal NMDAR-dependent synaptic plasticity, dendritic morphology and spatial memory. In line with the hypothesis of a hypofunction of NMDARs in the pathogenesis of schizophrenia, it has been shown that increased d-aspartate levels also improve brain connectivity, produce corticostriatal adaptations resembling those observed after chronic haloperidol treatment, and protects against prepulse inhibition deficits and abnormal circuits activation induced by psychotomimetic drugs. In healthy humans, genetic variation predicting reduced expression of DDO in post-mortem prefrontal cortex is associated with greater prefrontal gray matter and activity during working memory. On the other side, evaluation of d-aspartate content in post-mortem patients with schizophrenia has shown a significant reduction of this d-amino acid in the prefrontal cortex and striatum. Generation of mouse models with reduced embryonic levels of d-aspartate may disclose unprecedented role for d-aspartate in developmental brain processes associated with vulnerability to psychotic-like symptoms

Electrophysiological analysis of the modulation of NMDA-receptors function by D-serine and glycine in the central nervous system

International audienceThe NMDA subtypes of glutamatergic receptors (NMDARs) are unusual in that their activation requires the binding of both glutamate and a co-agonist glycine or D-serine. Whereas glycine was first suggested to play such a role, it was later established that D-serine could serve as an endogenous co-agonist at different central synapses. We still do not know the exact nature of the endogenous co-agonist(s) of NMDARs and the function of the so-called glycine B site in many brain structures. We introduced few years ago the use of enzymes that specifically degrade either D-serine or glycine to decipher the influence of these amino acids on NMDA receptors function. The use of these enzymatic scavengers represents an invaluable technique for neurophysiologists investigating the neuromodulation of the glycine B site in the CNS. Here, we describe the proper way to manipulate these enzymes during electrophysiological recordings in acute brain slices and highlight the experimental tricks

Confocal imaging and tracking of the exocytotic routes for D-serine-mediated gliotransmission.

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

Excitatory drive of cortical fast-spiking GABAergic interneurons is set by D-serine acting on NMDA receptors

Abstract N-methyl-D-aspartate receptors (NMDARs) populate GABAergic interneurons, where they play a critical role in shaping circuit motifs and memory. However, we are largely ignoring whether and how NMDARs at GABAergic interneurons are gated by signals released in their surrounding microenvironment. Here we explore the dynamics of the co-agonist site occupancy by D-serine and glycine at glutamatergic synapses onto parvalbumin positive GABAergic interneurons in the adolescent prefrontal cortex, an area central to complex cognitive operation. We discovered that D-serine but not glycine is required for maintaining the activity of NMDAR at the GABAergic interneurons and that the identity of the co-agonist is not determined by the synaptic regime. Our study extends the physiological implications of D-serine in brain physiopathology by uncovering its control of inhibitory synaptic networks through NMDARs

A multimodal nanopipette-based imaging platform for exploring brain communication

International audienceN-methyl-D-aspartate receptors (NMDARs) are ionotropic glutamate receptors that are critical for multiple aspects of brain physiology and pathology. They are essential for neural coding and transducing specific patterns of synaptic activity into long-term structural and functional changes of synapses and, therefore, vital to cognitive processes. Conversely, malfunction of these receptors is linked to the etiology of brain disorders such as Alzheimer disease and schizophrenia, for which there is no treatment. Therefore, interpreting mechanisms associated in their regulation is very important, however some of the molecular mechanisms governing the functioning of NMDARs are still unknown due to the lack of suitable techniques [1-2]. We are in particular interested in measuring the concentrations of NMDARs' coagonists, glycine and D-Serine, with high spatial resolution in order to understand their function in physiological and possible pathological conditions [3-4].We propose the development of a multimodal tool based on the scanning ion conductance microscopy (SICM), a well established scanning probe microscopy technique that provides topographic information of biological samples and living tissues with high resolution [5]. Excitingly, in our imaging platform, the scanning probe will also be capable of measuring D-serine and glycine concentrations at synaptic and extra-synaptic nanodomains with a nanometer scale resolution, by means of an electrochemical enzymatic biosensors embedded in the tip of probe itself. The first step towards the employment of the platform for our purpose consisted of several tests to adapt the functioning of the setup for imaging two different cell types: neuron-like PC12 cells and MDCK cells. In parallel, first stages of the nano-pipette probes functionalization have been investigated to make it suitable for future inclusion of the enzymatic biosensor. Merging the imaging capabilities of the methodology and the advancements in tip engineering will allow to measure, in a network of mature neurons, the concentrations of the two co-agonists of NMDARs, while providing the topography of the synapses where these molecules are active.Bibliography1. Traynelis, S. F. et al. Glutamate Receptor Ion Channels: Structure, Regulation, and Function. Pharmacological Reviews 62, 405{496 (2010)2. Paoletti, P., Bellone, C. Zhou, Q. NMDA receptor subunit diversity: impact on receptor properties, synaptic plasticity and disease.Nature Reviews Neuroscience 14, 383-400 (2013)3. Mothet, J.-P., le Bail, M. Billard, J.-M. Time and space profiling of NMDA receptor co-agonist functions. Journal of Neurochemistry 135,210-225 (2015).4. Mothet, J.-P. et al. D-Serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate. receptor. Proceedings of the National Academy of Sciences 97, 4926-4931 (2000).5. Zhu, C., Huang, K., Siepser, N. P., Baker, Scanning Ion Conductance Microscopy, Chemical Reviews 121, 11726-11768 (2021)

Investigating brain d ‐serine: Advocacy for good practices

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