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

Alix is required for activity-dependent bulk endocytosis at brain synapses

In chemical synapses undergoing high frequency stimulation, vesicle components can be retrieved from the plasma membrane via a clathrin-independent process called activitydependent bulk endocytosis (ADBE). Alix (ALG-2-interacting protein X/PDCD6IP) is an adaptor protein binding to ESCRT and endophilin-A proteins which is required for clathrinindependent endocytosis in fibroblasts. Alix is expressed in neurons and concentrates at synapses during epileptic seizures. Here, we used cultured neurons to show that Alix is recruited to presynapses where it interacts with and concentrates endophilin-A during conditions triggering ADBE. Using Alix knockout (ko) neurons, we showed that this recruitment, which requires interaction with the calcium-binding protein ALG-2, is necessary for ADBE. We also found that presynaptic compartments of Alix ko hippocampi display subtle morphological defects compatible with flawed synaptic activity and plasticity detected electrophysiologically. Furthermore, mice lacking Alix in the forebrain undergo less seizures during kainate-induced status epilepticus and reduced propagation of the epileptiform activity. These results thus show that impairment of ADBE due to the lack of neuronal Alix leads to abnormal synaptic recovery during physiological or pathological repeated stimulations

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

VGLUT1 functions as a glutamate/proton exchanger with chloride channel activity in hippocampal glutamatergic synapses

Glutamate is the major excitatory transmitter in the vertebrate nervous system. To maintain synaptic efficacy, recycling synaptic vesicles (SV) are refilled with glutamate by vesicular glutamate transporters (VGLUTs). The dynamics and mechanism of glutamate uptake in intact neurons are still largely unknown. Here, we show by live-cell imaging with pH- and chloride-sensitive fluorescent probes in cultured hippocampal neurons of wild-type and VGLUT1-deficient mice that in SVs VGLUT functions as a glutamate/proton exchanger associated with a channel-like chloride conductance. After endocytosis most internalized Cl− is substituted by glutamate in an electrically, and presumably osmotically, neutral manner, and this process is driven by both the Cl− gradient itself and the proton motive force provided by the vacuolar H+-ATPase. Our results shed light on the transport mechanism of VGLUT under physiological conditions and provide a framework for how modulation of glutamate transport via Cl− and pH can change synaptic strength

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

Glutamate receptor activation triggers a calcium-dependent and SNARE protein-dependent release of the gliotransmitter D-serine

The gliotransmitter d-serine is released upon (S)-α-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

Evaluation de la télédéclaration pour estimer l’incidence de l’érythème migrant en Pays des Combrailles : étude Pilote

National audienceComme les manifestations précoces et tardives de la maladie de Lyme sont parfois difficiles à diagnostiquer avec certitude, une stratégie envisagée pour évaluer l’incidence de la maladie est d’enregistrer l’occurrence de l’Erythème Migrant (EM), une manifestation cutanée pathognomonique des premières phases de la maladie. L’objectif de cette étude était d’évaluer la faisabilité de la télédéclaration de l’EM pour estimer l’incidence de la maladie de Lyme. L’étude a été menée dans une zone rurale de 47 000 habitants, les Combrailles (Auvergne), connue pour héberger de nombreuses tiques Ixodes ricinus infectées par Borrelia burgdorferi. Une campagne d’information a été organisée à l’attention des habitants et des professionnels de santé. Ont été incluses dans l’étude les personnes ayant (i) envoyé une image d’une lésion cutanée suspecte à l’adresse mail et/ou au numéro de téléphone dédiés entre le 1er avril 2017 et le 31 mars 2018 et (ii) ayant répondu à un questionnaire. Deux médecins, infectiologue et dermatologue, du CHU de Clermont-Ferrand ont jugé la qualité de l’image et la probabilité d’un EM. Parallèlement, un questionnaire a été envoyé aux médecins généralistes et aux pharmaciens de la région afin d’estimer le nombre de cas vus en consultations sur la même période. Au total, 113 images ont été reçues (mails ou MMS). Parmi les déclarants, 73 étaient hors zone ou hors période d’étude et 9 n’ont pas répondu au questionnaire. Sur les 31 personnes ayant envoyé une photo dans la zone et la période d’étude, 24 ont répondu au questionnaire. Parmi elles, 5 personnes ont déclaré ne pas avoir de smartphone. Tous les participants sauf 2 ont envoyé facilement les photos, toutefois dans 9 cas sur 31 l’envoi a été effectué par un tiers (famille, pharmacien, médecin, infirmier). Dix-huit sur 31 (58%) étaient des femmes. L’âge médian était de 51,5 ans [35-58]. La qualité des photos variait de très bonne (71%), bonne (23%) à médiocre (6%). Cinq images (16%) ont été évaluées comme érythème migrant probable, 7 (23%) possible, 7 (23%) peu probable et dans 12 cas (38%), le diagnostic d’EM a été rejeté. La majorité des déclarations ont été recensée pendant les mois de mai à aout, que ce soit par la population, les médecins ou les pharmaciens. L’estimation de l’incidence des EM par télédéclaration est un outil prometteur. Toutefois cette étude pilote montre des limites : nécessité d’une campagne de sensibilisation préalable (affiches, flyers, presse, radio, réunion d’informations pour médecins, pharmaciens et grand public), intervention impérative de médecins pour la collecte des données cliniques et l’interprétation des images permettant d’appuyer l’hypothèse d’un EM