Mauricio Dos-Santos-Pereira

Leonardo Acuña

Sabah Hamadat

Jeremy Rocca

Florencia González

Rosana Chehín

Julia Sepulveda-Diaz

Elaine Del-Bel

Rita Raisman-Vozari

Patrick Pierre Michel

Microglial glutamate release evoked by -synuclein aggregates is prevented by dopamine

Keywords: 9, Main points: 27, Text: 6181, References: 2404, Figure legends: 1041 -Synuclein, Glutamate, Microglia, Neuroinflammation, Parkinson

When activated, microglial cells have the potential not only to secrete typical proinflammatory mediators but also to release the neurotransmitter glutamate in amounts that may promote excitotoxicity. Here, we wished to determine the potential of the Parkinson's disease (PD) protein α-Synuclein (αS) to stimulate glutamate release using cultures of purified microglial cells. We established that glutamate release was robustly increased when microglial cultures were treated with fibrillary aggregates of αS but not with the native monomeric protein. Promotion of microglial glutamate release by αS aggregates (αSa) required concomitant engagement of TLR2 and P2X7 receptors. Downstream to cell surface receptors, the release process was mediated by activation of a signaling cascade sequentially involving phosphoinositide 3-kinase (PI3K) and NADPH oxidase, a reactive oxygen species-producing enzyme. Inhibition of the Xcantiporter, a plasma membrane exchange system that imports extracellular L-cystine and exports intracellular glutamate, prevented the release of glutamate induced by αSa, indicating that system Xc-was the final effector element in the release process downstream to NADPH oxidase activation. Of interest, the stimulation of glutamate release by αSa was abrogated by dopamine through an antioxidant effect requiring D1 dopamine receptor activation and PI3K inhibition. Altogether, present data suggest that the activation of microglial cells by αSa may possibly result in a toxic build-up of extracellular glutamate contributing to excitotoxic stress in PD. The deficit in dopamine that characterizes this disorder may further aggravate this process in a vicious circle mechanism.

Introduction

Parkinson disease (PD) is a multifactorial neurodegenerative disorder primarily characterized by a loss of midbrain substantia nigra dopamine (DA) neurons and ensuing motor deficits [START_REF] Mullin | Pathogenic mechanisms of neurodegeneration in Parkinson disease[END_REF][START_REF] Michel | Understanding Dopaminergic Cell Death Pathways in Parkinson Disease[END_REF]. Neuronal cell loss is probably the result of intrinsic mechanisms that affect neuronal function and ultimately neuronal viability. It is believed, however, that neurodegeneration is exacerbated by sustained inflammatory processes that implicate microglial cells [START_REF] Hirsch | The role of glial reaction and inflammation in Parkinson's disease[END_REF]McGeer and Mc Geer, 2008), the resident immune cells of the brain [START_REF] Ginhoux | Fate mapping analysis reveals that adult microglia derive from primitive macrophages[END_REF][START_REF] Bohlen | Diverse Requirements for Microglial Survival, Specification, and Function Revealed by Defined-Medium Cultures[END_REF]. Microglial activation in PD may not simply occur as a consequence of neuronal damage as originally thought but also as an intrinsic component of the disease process [START_REF] Mouton-Liger | Parkin deficiency modulates NLRP3 inflammasome activation by attenuating an A20-dependent negative feedback loop[END_REF].

Microglial cell activation was demonstrated in post-mortem tissue [START_REF] Hirsch | The role of glial reaction and inflammation in Parkinson's disease[END_REF]McGeer and Mc Geer, 2008) and in living patients diagnosed with PD, using positron emission tomography imaging techniques [START_REF] Ouchi | Neuroinflammation in the living brain of Parkinson's disease[END_REF]; Bartels et al. 2010). The contribution of inflammatory processes in PD progression is also indirectly supported by epidemiological studies showing that the PD risk is reduced in long-term users of some nonsteroidal anti-inflammatory medications [START_REF] Gagne | Anti-inflammatory drugs and risk of Parkinson disease: a meta-analysis[END_REF][START_REF] Rees | Nonsteroidal anti-inflammatory drugs as disease-modifying agents for Parkinson's disease: evidence from observational studies[END_REF].

A number of studies suggest that the aggregation process which affects the synaptic protein α-Synuclein (αS), could represent a potential trigger for microglial cell activation [START_REF] Zhang | Aggregated -synuclein activates microglia: a process leading to disease progression in Parkinson's disease[END_REF][START_REF] Couch | The acute inflammatory response to intranigral α-synuclein differs significantly from intranigral lipopolysaccharide and is exacerbated by peripheral inflammation[END_REF][START_REF] Lema Tomé | Inflammation and α-synuclein's prion-like behavior in Parkinson's disease -is there a link[END_REF]. Microglial activation may result from the presence of pathological S aggregates that diffuse and propagate in the brain parenchyma of PD patients [START_REF] Emmanouilidou | Assessment of α-synuclein secretion in mouse and human brain parenchyma[END_REF][START_REF] Dijkstra | Stage-dependent nigral neuronal loss in incidental Lewy body and Parkinson's disease[END_REF][START_REF] Bernis | Prion-like propagation of human brain-derived -synuclein in transgenic mice expressing human wild-type -synuclein[END_REF] to finally form cytoplasmic inclusions, termed Lewy bodies, and abnormal neuritic depositions, termed Lewy neurites [START_REF] Braak | Neuropathological Staging of Brain Pathology in Sporadic Parkinson's disease: Separating the Wheat from the Chaff[END_REF]. Consistent with this hypothesis, the extent of microglial activation estimated by HLA-DR immunostaining was reported to be closely correlated with the amount of αS deposited in the brain of PD patients [START_REF] Croisier | Microglial inflammation in the parkinsonian substantia nigra: relationship to -synuclein deposition[END_REF].

Here, our specific aim was to evaluate the pro-inflammatory potential of amyloid fibrils of S, using glutamate release as a marker of the activation state of these cells.

Our interest in this marker lies in the fact that activated microglial cells can release glutamate in amounts that have the propensity to promote excitotoxic stress [START_REF] Mesci | System xC-is a mediator of microglial function and its deletion slows symptoms in amyotrophic lateral sclerosis mice[END_REF], a glutamate-receptor-mediated process thought to contribute to PD neurodegeneration [START_REF] Wallace | Survival of midbrain dopaminergic cells after lesion or deep brain stimulation of the subthalamic nucleus in MPTP-treated monkeys[END_REF][START_REF] Ambrosi | A further update on the role of excitotoxicity in the pathogenesis of Parkinson's disease[END_REF].

So, we established a model system of pure microglial cells in culture and used these cultures to evaluate the impact of S aggregates (Sa) on glutamate release. We found that Sa had a strong stimulatory effect on glutamate release through a sequence of signaling events that ultimately lead to activation of the cystine/glutamate antiporter system Xc -. Most interestingly, the stimulatory effect of Sa on glutamate release was totally suppressed by the neurotransmitter DA which operated through activation of D1 DA receptors.

Materials and methods

Pharmacological and cell culture reagents

Lipopolysaccharide (LPS; Escherichia coli strain O26:B6; #L8274), 4',6-Diamidino-2-PhenyIindole (DAPI), sulfasalazine (Sulfa; #S0883); benzoylbenzoyl-ATP (BzATP; #B6396), dopamine (DA) hydrochloride (#H8502), SKF-81,297 hydrobromide (SKF; #S179), 2-bromo-α-ergocryptine methane sulfonate salt (ERG; #B2134), forskolin (FK; #F6886), trolox (TROL; #238813) were all purchased from Sigma Aldrich (L'Isle d'Abeau Chesnes, France). JNJ-47965567 (JNJ; #5299), LY294002 hydrochloride (LY; #1130) and diphenyliodonium (DPI; #0504) were from Tocris Bioscience. Apocynin (APO; #4663) was purchased from R&D Systems Europe. The Novex TNF mouse ELISA kit was from Thermofischer Scientific (Illkirch, France). The synthetic triacylated lipoprotein Pam3CSK4 (#vac-pms) was from Invivogen (Toulouse, France). Dulbecco's modified Eagle's medium (DMEM), DMEM/F-12 nutrient mixture, EDTA (2 mM)-Trypsin (0.05%), the Amplex Red Glutamic Acid/Glutamate Oxidase Assay Kit (#A12221) and the CellROX Deep Red Reagent were all purchased from Invitrogen Life Technologies (Saint Aubin, France). N5 medium which is not commercially available was prepared using reagents from Sigma-Aldrich [START_REF] Kaufman | Serum factor supporting long-term survival of rat central neurons in culture[END_REF]. Fetal bovine serum (FCS) and horse serum were obtained from Biowest LLC (Eurobio, Les Ulis, France) and Sigma Aldrich, respectively. Radioactive [ 14 C]-L-Cystine was obtained from Perkin Elmer (Courtaboeuf, France).

Primary antibodies

The MAC-1/CD-11b (clone M1/70.15, Cat# MCA74GA, RRID: AB_324660) antibody was from Bio-Rad/AbD Serotec (Oxford, UK) and the rabbit anti-ionized calcium binding adaptor molecule-1 (Iba-1; Cat# 019-19741, RRID: AB_839504) antibody from Wako Chemicals (Neuss, Germany). The rabbit polyclonal p47-phox antibody (Cat# sc-14015, RRID: AB_2150289) was from Cliniscience (Nanterre, France). The rabbit monoclonal antibody against AKT phosphorylated at Ser473 (p-AKT; Cat #4060, RRID: AB_2315049) and the rabbit polyclonal antibody against total AKT (Cat #9272, RRID: AB_329827) were both purchased from Cell Signaling Technology (Leiden, Netherlands). The rabbit polyclonal antibody against human αS (Cat# BML-SA3400, RRID: AB_2192956) was from Enzo Life Sciences (Lyon, France) and the polyclonal rabbit GADPH antibody (Cat# G9545, RRID: AB_796208) from Sigma Aldrich. The mouse monoclonal anti-Toll-like receptor (TLR)2-IgG antibody (Cat# mabg-mtlr2, RRID:AB_11125339), an antagonistic antibody for TLR2 was from Invivogen.

Production of glial cell cultures

Animal care and handling: Animals were housed, handled, and cared for in strict accordance with the European Union Council Directives (2010/63/EU). The protocol was approved by the Committee on the Ethics of Animal Experiments Charles Darwin n°5.

Coating procedures: When needed, bottom surface areas of culture vessels were covered with 1 mg/ml polyethyleimine (PEI; weight average molar mass 750,000; #P3143, Sigma Aldrich) diluted in a pH 8.3 borate buffer solution (40 mM) [START_REF] Rousseau | The iron-binding protein lactoferrin protects vulnerable dopamine neurons from degeneration by preserving mitochondrial calcium homeostasis[END_REF]. After at least 1-2 h at 37°C, culture vessels were washed 4 times with Dulbecco's phosphate buffered saline medium (PBS) before application of culture medium and cell seeding. We also used laminin (#11243217001, Sigma Aldrich) diluted in PBS as a coating [START_REF] Sepulveda-Diaz | A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro[END_REF] for experiments made with astrocytes.

Microglial cell cultures: Microglial cell were isolated from the brain of newborn mouse pups (post-natal day 1) using a simple culture procedure that allows spontaneous and selective isolation of these cells in bulk quantities [START_REF] Sepulveda-Diaz | A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro[END_REF]. Briefly, the isolation was performed by plating mechanically dissociated brain cells in PEI-coated Corning T-75 flasks (Sigma Aldrich) containing DMEM supplemented with 10% FCS and antibiotics. After a single medium change for removing cellular debris at day 2 in vitro, the cultures were kept as such for 14-16 more days until completion of microglial cell isolation. When needed, purified microglial cell cultures were maintained longer in culture flasks by completing the cultivation medium with a small aliquot of fresh medium. To produce subcultures, microglial cells were recovered by trypsin proteolysis using an EDTA (2 mM)-trypsin (0.05%) solution and seeded in PEI-coated 48 multi-well plates at a density of about 100,000 cells. The cultures were then maintained in N5 medium, a cerebrospinal fluid-like medium [START_REF] Kaufman | Serum factor supporting long-term survival of rat central neurons in culture[END_REF]. This medium was supplemented with 5% horse serum, 0.5% FCS, 5 mM glucose and 100 µM glycine.

Cultures were maintained at 37°C under a humidified atmosphere of 95% air and 5% CO2. These cultures were virtually free of astrocytes.

Astrocyte cultures: To produce cultures enriched in astrocytes, we used a protocol of isolation that differed to that described previously for microglial cells, in that the coating was laminin and the culture medium DMEM/F-12 nutrient mixture supplemented with 10% FCS and antibiotics. The growth of these cultures was stopped after 2 weeks, i.e., at a stage where >90% of the cultured cells are astrocytes. To produce subcultures, adherent cells were recovered by trypsin proteolysis as described previously for microglial cells and seeded at a density of about 100,000 cells in Nunc 48 multi-well plates coated with laminin and filled with N5 medium supplemented with serum.

Experiments of stimulation were performed in confluent astrocyte cultures.

Purification of recombinant αS

Human αS was expressed in Escherichia coli using the pT7-7 expression plasmid and purified as described [START_REF] Hoyer | Dependence of -synuclein aggregate morphology on solution conditions[END_REF][START_REF] Kaylor | Characterization of oligomeric intermediates in -synuclein fibrillation: FRET studies of Y125W/Y133F/Y136F -synuclein[END_REF][START_REF] González-Lizárraga | Repurposing doxycycline for synucleinopathies: remodelling of α-synuclein oligomers towards non-toxic parallel beta-sheet structured species[END_REF]. The recombinant protein was recovered in 20 mM HEPES, 150 mM NaCl, pH 7.4 and its purity assessed by SDS-PAGE electrophoresis. Protein samples were then applied to a Pierce spin column (#88275; Thermoscientific) to remove endotoxins potentially present as contaminants. The endotoxin concentration in protein eluates was constantly below the detection threshold (0.1 EU/ml) of the chromogenic Limulus Amebocyte Lysate assay (#88282; Thermoscientific). Protein solutions were then filtered and centrifuged at 12,000× g for 30 min and the protein content of the supernatant measured at 280 nm with a Nanodrop 8000 Spectrophotometer (Thermofischer Scientific) using an extinction coefficient of 5600 cm -1 M -1 . The protein concentration was finally adjusted to appropriate concentrations.

Preparation of αS aggregates

To produce aggregates, recombinant αS samples were incubated at 37°C in an orbital shaker (Thermomixer comfort; Eppendorf; Montesson, France) set at 600 rpm. After 96 h of incubation, protein aggregates were harvested, then sonicated for 2 min using a Branson B3510-DTH ultrasonic bath (VWR International, Fontenay sous Bois, France), and kept at -20°C until further use as previously described [START_REF] González-Lizárraga | Repurposing doxycycline for synucleinopathies: remodelling of α-synuclein oligomers towards non-toxic parallel beta-sheet structured species[END_REF]. The formation of amyloid-type cross-beta structures was confirmed with the Thioflavin-T fluorescence assay [START_REF] Levine | Quantification of beta-sheet amyloid fibril structures with thioflavin T[END_REF]. The presence of amyloid fibrils was was further demonstrated by Transmission Electron Microscopy (TEM) using samples (5 µl) that were deposited on carbon Formvar coated grids (Electron Microscopy Sciences, Hatfield, PA; FF100-Cu, 100 Mesh). After enhancing the contrast with 2% uranyl acetate in water for 5 min at room temperature, the preparations were examined using a Hitachi HT7700 electron microscope (Elexience, Verrières-le-Buisson) operating at 70 kV as described [START_REF] Ndour | Measuring the Plasmodium falciparum HRP2 protein in blood from artesunate-treated malaria patients predicts post-artesunate delayed hemolysis[END_REF].

Protein detection by immunofluorescence

Cultures were fixed with 4% formaldehyde in PBS (20 min, room temperature), washed once with PBS, and then incubated with antibodies against MAC-1 (1:1000 in PBS for 72 h), Iba-1 (1:500 in PBS-Triton 0.2% overnight), αS (1:500 in PBS or PBS-triton 0.05% overnight), or p47-phox/NADPH oxidase (NOX)2 (1:50 in PBS-triton 0.05%, overnight). When MAC1/Iba-1 double immunostaining was performed, MAC-1 and Iba-1 were detected, sequentially. When MAC-1 was detected along with αSa, αS was used either diluted in PBS or in PBS-triton 0.05%. Suitable Alexa-Fluor 488 and Alexa-Fluor 555 conjugated secondary antibodies (Invitrogen) were used at a concentration of 1:250. Nuclei of labeled cells were counterstained with 4',6-diamidino-2-phenylindole (DAPI; 5 µg/ml for 2 min). Fluorescent images were acquired using a Nikon TE 2000 inverted microscope (Nikon, Tokyo, Japan) equipped with an ORCA-ER digital cooled camera and the HCI imaging software. When needed, changes in immunofluorescent signal intensities were quantified at the cellular level using protocols described previously [START_REF] Sepulveda-Diaz | A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro[END_REF].

Counts of DAPI + nuclei

Numbers of DAPI + nuclei were estimated by automated counting using an Arrayscan XTi workstation equipped with the HCS Studio 2.0 Software (ThermoScientific, Courtaboeuf, France).

Western immunoblotting

Microglial cells were first pretreated with pharmacological reagents and 2 h later with αSa. After 30 min (for p-AKT) and 24 h (for total AKT or GADPH) of αSa stimulation, cells were processed using a protocol described earlier by Santa-Cecília and colleagues (2016). Briefly, adherent cells were treated with a M-PER buffer (#78501; Invitrogen) containing a protease and phosphatase inhibitor cocktail (#78440; Invitrogen) and aliquots of lysates were recovered for protein quantification with a Nanodrop 8000 Spectrophotometer (Thermofischer Scientific). Equal amounts of protein lysates were then resolved on a 10% polyacrylamide gel (SDS-PAGE) before transfer to polyvinylidene difluoride membranes. Membranes incubated with primary antibodies against p-AKT (1:1000; 30 min.), AKT (1:1000; overnight) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:2000; 2 h) were washed with a Tris-buffered saline Tween-20 solution and then incubated with a rabbit secondary IRDye antibody (LI-COR Biosciences, Lincoln, NE, USA). Blots were imaged using a LI-COR Odyssey infrared imaging system (LI-COR Biosciences) and quantitative analysis of stained bands was performed with the ImageJ software (dos-Santos [START_REF] Dos Santos Pereira | Long Withdrawal of Methylphenidate Induces a Differential Response of the Dopaminergic System and Increases Sensitivity to Cocaine in the Prefrontal Cortex of Spontaneously Hypertensive Rats[END_REF].

Glutamate assay

Glutamate was assayed using the Amplex Red Glutamic Acid/Glutamate Oxidase Kit (#A12221; Invitrogen) according to the manufacturer's instructions. The quantification was carried out with 25 µl of culture medium and the fluorescent reaction product resulting from glutamic acid oxidation was quantified using a SpectraMax M4 microplate reader (Molecular Devices, Sunnyvale, CA).

Measurement of [ 14 C]-Cystine uptake

Cystine transport was measured based on a protocol described by Tomi and colleagues (2002). Briefly, microglial cells exposed to test treatments for 2h at 37°C were then incubated for the next 30 min with 8 µM [ 14 C]-Cystine. The uptake was terminated by washing the cultures 3 times with ice-cold PBS. The radioactivity accumulated by cultured cells was released by treatment with distilled water and quantified by liquid scintillation counting.

Quantification of intracellular ROS levels

Intracellular reactive oxygen species (ROS) were assessed using the membrane permeable CellROX Deep Red Reagent (Invitrogen Life Technologies), as described elsewhere [START_REF] Sepulveda-Diaz | A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro[END_REF].

Statistical analysis

Experimental values expressed as mean ±S.E.M. were derived from at least two sets of independent experiments. Statistical analyses were performed with the SPSS software for Windows (version 20). All pairwise comparisons were made by one-way ANOVA followed by the Student-Newman-Keuls post hoc test.

Results αSa stimulate glutamate release in microglial cell cultures

We measured the impact that a 24-hour treatment with monomeric αS (αSm) (70 µg/ml) or with αSa (15 and 70 µg/ml) had on the release of glutamate in PEI-isolated microglial cell cultures, using LPS (10ng/ml) as a reference inflammogen [START_REF] Lu | LPS/TLR4 signal transduction pathway[END_REF].

A treatment with 15µg/ml αSa caused a small but non-significant increase in glutamate release. With 70µg/ml αSa, however, the release of glutamate was robustly and significantly increased (p < 0.0001 vs control) (Figure 1a).The impact of 70 µg/ml αSa on glutamate release was roughly equivalent to that produced by 10 ng/ml LPS (p < 0.0001 vs control). Importantly, a treatment of microglial cell cultures with 70 µg/ml αSm did not affect glutamate release, indicating that non-aggregated monomeric forms of αS had no influence on this process. TEM images shown in Figure 1b confirmed that amyloid fibrils were present in αS shaken samples, only (lower image).

Astroglial cells which are also known to promote inflammatory-type reactions during neurodegeneration [START_REF] Liddelow | Reactive Astrocytes: Production, Function, and Therapeutic Potential[END_REF], failed to release glutamate in response to αSa (70 µg/ml) (Figure 1c). A similar result was found when astrocytes were challenged with LPS (10 ng/ml).

To determine to what extent increases in glutamate release induced by αSa (70 µg/ml) or LPS (10 ng/ml) were related to changes in microglial cell numbers, we counted the number of DAPI + nuclei in cultures receiving or not these treatments (Figure 1d). We found that the number of DAPI + nuclei was slightly augmented by about 1.25-and 1.15fold in αSa (70 µg/ml)-and LPS (10ng/ml)-treated cultures, respectively (p < 0.001 and p < 0.05 vs control).

We also evaluated the impact of Sa on microglial cells using other inflammation markers. Sa (70 µg/ml) stimulated the release of the prototypical inflammatory cytokine TNF- with similar efficacy as LPS (10 ng/ml) (p<0.001 vs control) (Figure 1e). Sa also strongly enhanced the expression of the microglial activation marker Iba-1 in CD11b + cells (Figure 1f), indicating that the increase in glutamate release induced by the fibrillary forms of the protein was reflecting a more generalized inflammatory state in microglial cells. αSa stimulate glutamate release in microglial cells through a receptor-mediated process.

We found that αSa were frequently co-localized with the immunofluorescence signal for the cell surface integrin CD11b, when the antibody against human αS was used without permeabilization (Figure 2a). Aggregates were virtually absent in microglial cell bodies in these conditions (Figure 2a). Intracellular aggregates of αS remained also scarce after permeabilization (unshown data), suggesting that the intrinsic phagocytocytic activity of microglial cells was reduced in present culture conditions or alternatively that our antibody did not recognize particules that had been phagocytized.

The presence of aggregates close to plasma membranes was an indication that the induction of glutamate release by αSa was possibly mediated by activation of microglial cell membrane receptors. To address this point, microglial cells were exposed to αSa (70 µg/ml) in the presence or not of MAb mTLR2 (2.5 µg/ml), an antagonistic antibody for TLR2 receptors [START_REF] Chen | A53T human α-synuclein overexpression in transgenic mice induces pervasive mitochondria macroautophagy defects preceding dopamine neuron degeneration[END_REF]. In the same setting, we also tested the impact of JNJ (20 µM), a synthetic antagonist for purinergic P2X7 receptors [START_REF] Letavic | Synthesis and Pharmacological Characterization of Two Novel, Brain Penetrating P2X7 Antagonists[END_REF]. Figure 2b shows that glutamate release induced by αSa was markedly reduced by each of the receptor antagonists JNJ and MAb mTLR2 (p < 0.0001). When MAb mTLR2 and JNJ were added concurrently to αSa-treated cultures, glutamate release returned below control values (p < 0.0001), indicating that both TLR2 and P2X7 receptors contributed to the release of the neurotransmitter in this setting. Note that in the absence αSa, the basal release of glutamate was almost totally abolished in cultures treated with the two receptor antagonists.

Consistent with this set of results, we found that specific agonists for TLR2 and P2X7 receptors, Pam3CSK4 (1 µg/ml) [START_REF] St Paul | Toll-like receptor ligands induce the expression of interferon-gamma and interleukin-17 in chicken CD4 + T cells[END_REF] and BzATP (500 µM) [START_REF] Young | Amino acid residues in the P2X7 receptor that mediate differential sensitivity to ATP and BzATP[END_REF], respectively, led to robust stimulation of glutamate release in microglial cell cultures (p < 0.0001 vs control) (Figure 2c). As expected, MAb mTLR2 (2.5 µg/ml) largely antagonized the induction of glutamate release elicited by Pam3CSK4 (p < 0.01 vs PAM) whereas JNJ (20 µM) reduced that elicited by BzATP (p < 0.001 vs BzATP). When Pam3CSK4 and BzATP were added together to the cultures, the increase in glutamate release appeared to be stronger than with each individual treatment (p < 0.0001 vs control). In this situation, a combination of MAb mTLR2 and JNJ was required to produce a substantial reduction in glutamate release (p < 0.0001 vs BzATP + PAM).

Glutamate release induced by αSa in microglial cells occurs through activation of the

Xc -antiporter system

Next, we tested the possibility that αSa could stimulate glutamate release by activating the antiporter system Xc -, a membrane protein that imports into the cells the amino acid L-cystine, the oxidized form of cysteine, and exports L-glutamate in a 1:1 ratio [START_REF] Lewerenz | The cystine/glutamate antiporter system Xc-in health and disease: from molecular mechanisms to novel therapeutic opportunities[END_REF][START_REF] Soria | Cystine/glutamate antiporter blockage induces myelin degeneration[END_REF]. To test this possibility, we studied the impact of the Xc -inhibitor Sulfa [START_REF] Shukla | Inhibition of xc⁻ transporter-mediated cystine uptake by sulfasalazine analogs[END_REF] on the release of glutamate induced by αSa. Figure 3a shows that the induction of glutamate release by αSa (70 µg/ml; p < 0.0001 vs control) was completely antagonized by Sulfa (50 µM; p < 0.0001 vs αSa). Note that basal glutamate release was also totally suppressed by Sulfa in the absence of αSa (p < 0.0001 vs control), suggesting that the spontaneous release of glutamate resulted from basal activation of the Xc -system.

To confirm the implication of this transport system in the effect of αSa, we measured the uptake of [ 14 C]-L-cystine in the same experimental setting. Because of technical constraints imposed by the use of this radiolabeled compound, the uptake was assessed for only 30 min in cultures previously exposed for 2 h to test treatments. Figure 3b shows that the accumulation of [ 14 C]-cystine was strongly elevated in microglial cells that were exposed to αSa (p < 0.0001 vs control). Sulfa led to substantial reduction of this increase (p < 0.0001 vs αSa), confirming that aggregated forms of αS had the capacity to stimulate the Xc -antiporter system in microglial cells. Note that basal [ 14 C]-cystine uptake was also significantly decreased by Sulfa in control cultures (p < 0.01 vs control).

Glutamate release induced by αSa in microglial cells results from a rise in intracellular oxidative stress.

We next tested whether activation of the Xc -system and ensuing stimulation of glutamate release were due to a rise of intracellular oxidative stress in microglial cells.

For that, we tested the impact of various antioxidants on glutamate release in αSatreated cultures. Figure 4a shows that the increase in glutamate release induced by 70µg/ml αSa was largely reduced by DPI (1 µM) and totally abolished by APO (300 µM), i.e., two compounds that operate as inhibitors of the superoxide producing enzyme NOX [START_REF] Lelli | The NADPH oxidase Nox2 regulates VEGFR1/CSF-1Rmediated microglial chemotaxis and promotes early postnatal infiltration of phagocytes in the subventricular zone of the mouse cerebral cortex[END_REF]) (APO: p < 0.0001 vs αSa; DPI: p < 0.001 vs αSa). The cell permeable analog of vitamin E, TROL (10 µM) was found as efficient as APO in the present setting (p < 0.0001 vs αSa).

So, to further confirm that oxidative stress was implicated in the stimulatory effects that αSa exert on glutamate release in microglial cells, we measured intracellular ROS production using the membrane permeable fluorogenic probe CellROX Deep Red Reagent [START_REF] Sepulveda-Diaz | A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro[END_REF]. As expected ROS production was enhanced by 70µg/ml αSa (p < 0.0001 vs control) in microglial cells (figure 4b,c) and prevented by APO, DPI and TROL (APO, DPI, TROL: p < 0.0001 vs αSa), i.e., treatments reducing glutamate release in αSa-treated cultures (Figure 4b,c).

The neurotransmitter dopamine prevents glutamate release induced by αSa in microglial cells through D1 receptor activation.

Next, we were interested in studying whether the neurotransmitter DA had an impact on the release of glutamate induced by αSa in microglial cell cultures. More specifically, we found that DA (10 µM) totally abolished the stimulatory action of αSa (70 µg/ml) on glutamate release (p < 0.0001 vs αSa) (figure 5a). Note that DA also suppressed basal glutamate release under control conditions (p < 0.0001 vs control).

We wished to further characterize the inhibitory action of DA on glutamate release induced by αSa in microglial cells. For that we tested the impact that specific D1 and D2

DA receptor agonists could have on this process. D1 receptors being most generally coupled to Gs and cAMP production [START_REF] Beaulieu | The physiology, signaling, and pharmacology of dopamine receptors[END_REF], we also tested the impact of the adenylate cyclase activator FK on glutamate release. LY (2.5 µM), an inhibitor of phosphoinositide 3-kinase (PI3K) was also tested in this experimental context (figure 5b). We found that the D1 DA receptor agonist SKF-81,297 (10 µM) was able to mimic the inhibitory action of DA on glutamate release (p < 0.0001 vs αSa) whereas the D2 agonist ERG (10 µM) failed to do so. We also established that FK (10 µM) led to substantial but only partial reduction of glutamate release in response to αSa exposure (p < 0.001 vs αSa) whereas LY (2.5 µM) had a strong inhibitory action under the same conditions (p < 0.0001 vs αSa). This last observation prompted us to study expression levels of p-AKT, the enzymatic product of PI3K, using a Western blotting approach. In particular, we compared conditions in which αSa was applied alone or in the presence of DA, FK or LY to microglial cell cultures (Figure 5c). Quantitative Western blot analysis revealed that the expression of p-AKT was induced by αSa (p < 0.05 vs control) and this effect was prevented by either DA (p < 0.001 vs αSa) or FK (p < 0.001 vs αSa) (figure 5d). As expected, LY also robustly inhibited p-AKT expression in microglial cell cultures exposed to αSa (p < 0.0001 vs αSa).

Dopamine prevents glutamate release induced by αSa in microglial cells through an antioxidant effect.

We wished to determine whether the reduction in glutamate release produced by DA in αSa-treated microglial cultures was possibly due to an antioxidant effect of the neurotransmitter. To do that, we measured intracellular ROS production in microglial cell cultures exposed to 70 µg/ml αSa in the presence or not of 10 µM DA (Figure 6a,b).

In the same setting, we also tested the impact of the adenylate cyclase activator FK (10 µM) and that of the PI3K inhibitor LY (2.5 µM) on ROS production (Figure 6a,b). Most notably, we found that the increase in intracellular ROS production induced by αSa was totally abolished by DA (10 µM) (p < 0.0001 vs αSa). Note that DA also reduced ROS production below control levels in the absence of αSa (p < 0.01 vs control). Interestingly, ROS produced by αSa were also reduced by the PI3K inhibitor LY (2.5 µM) (p < 0.01 vs αSa) and the adenylate cyclase activator FK (10 µM) (p < 0.05 vs αSa).

Finally, we tested the possibility that DA could prevent αSa-mediated ROS production by inhibiting the expression of NOX2, the NOX isoform that is most abundant in microglia cells [START_REF] Bedard | The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[END_REF]2007). For that we measured cellular expression levels of the NOX2 p47phox subunit in cultures exposed to αSa in the presence or not of DA or FK.

We also tested the impact of LY in this setting (Figure 6c,d). We established that p47phox expression was induced in αSa-treated microglial cultures (p < 0.0001 vs control) and that this induction was prevented when DA (10 µM) was added concomitantly to the cultures (p < 0.0001 vs αSa). Cyclic AMP elevation with FK (10 µM) caused a reduction in the induction of p47phox elicited by αSa (p < 0.05 vs αSa). Meanwhile, p47phox returned to basal levels in cultures exposed to αSa in the presence of LY (2.5 µM) (p < 0.0001 vs αSa). Overall, these data suggest that DA prevented PI3K-dependent activation of NOX2 by αSa. All these results are summarized in figure 7.

Discussion

Using a model system of post-natal microglial cells in culture, we established that aggregated (but not monomeric) forms of S had the capacity to robustly stimulate glutamate release through a mechanism requiring concomitant activation of TLR2 and P2X7 receptors and sequential stimulation of the PI3K and NOX enzymes. The Xc - antiporter system appeared to be the final downstream effector for Sa-mediated glutamate release. Interestingly, the effects of Sa on glutamate release were antagonized by the neurotransmitter DA through a mechanism that involves D1 DA receptors.

Aggregated but not monomeric forms of αS stimulate glutamate release in microglial cell cultures

The aggregation process of the synaptic protein αS represents one of the key pathological events in PD. It has been suggested that pathological forms of S that diffuse and propagate in the brain parenchyma [START_REF] Dijkstra | Stage-dependent nigral neuronal loss in incidental Lewy body and Parkinson's disease[END_REF][START_REF] Bernis | Prion-like propagation of human brain-derived -synuclein in transgenic mice expressing human wild-type -synuclein[END_REF] may contribute to disease progression by favoring inflammatory-type reactions mediated by microglial cells [START_REF] Couch | The acute inflammatory response to intranigral α-synuclein differs significantly from intranigral lipopolysaccharide and is exacerbated by peripheral inflammation[END_REF]. Here, we wished to evaluate the proinflammatory potential of amyloid fibrils of S, using glutamate release as an activation marker of these cells. Our specific interest for this marker comes from the fact that glutamate has the potential to generate low-level excitotoxic insults [START_REF] Lavaur | The noble gas xenon provides protection and trophic stimulation to midbrain dopamine neurons[END_REF] that may aggravate neurodegeneration in PD [START_REF] Ambrosi | A further update on the role of excitotoxicity in the pathogenesis of Parkinson's disease[END_REF]. Using a model system of purified microglial cell cultures, we established that amyloid fibrils of S had the capacity to stimulate glutamate release in a concentrationdependent manner. We found that both TNF- secretion and cellular expression of the microglial activation marker Iba-1 were also robustly enhanced in the same cultures, indicating that the stimulatory effect of Sa on glutamate release reflected a more generalized inflammatory state of microglial cells. This is coherent with previous reports reporting on the strong pro-inflammatory potential of S fibrillary species [START_REF] Zhang | Aggregated -synuclein activates microglia: a process leading to disease progression in Parkinson's disease[END_REF][START_REF] Wang | α-Synuclein, a chemoattractant, directs microglial migration via H2O2-dependent Lyn phosphorylation[END_REF][START_REF] Brück | Glia and -synuclein in neurodegeneration: A complex interaction[END_REF]. More precisely, we showed that 70 µg/ml Sa had an impact on glutamate release that was equivalent to that of 10 ng/ml of LPS, a cell-wall component that operates as a protypical inflammogen for microglial cells [START_REF] Barger | Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation[END_REF][START_REF] Sepulveda-Diaz | A simplified approach for efficient isolation of functional microglial cells: Application for modeling neuroinflammatory responses in vitro[END_REF]. Note that in some culture settings, toxic effects of aggregated forms of S have been reported at much lower concentrations [START_REF] Zhang | Aggregated -synuclein activates microglia: a process leading to disease progression in Parkinson's disease[END_REF][START_REF] Kim | Neuron-released oligomeric α-synuclein is an endogenous agonist of TLR2 for paracrine activation of microglia[END_REF]. Concentrations comparable to those used by us in the present study were, however, required in other paradigms [START_REF] Hoffmann | -synuclein activates BV2 microglia dependent on its aggregation state[END_REF][START_REF] Bussi | Autophagy down regulates pro-inflammatory mediators in BV2 microglial cells and rescues both LPS and alpha-synuclein induced neuronal cell death[END_REF]. We have no definite explanation for such differences, which may be attributable to different culture conditions and various procedures used to generate aggregated species of S.

At variance to Sa, Sm were totally ineffective in promoting glutamate release in microglial cells in the same experimental setting. These observations are coherent with reports showing that the inflammatory potential of S is dependent on the aggregation state of the protein [START_REF] Hoffmann | -synuclein activates BV2 microglia dependent on its aggregation state[END_REF]. Incidentally, the absence of effect of Sm confirms indirectly that the human recombinant S that we prepared for this study was free of bacterial contaminants susceptible to induce glutamate release in microglial cells [START_REF] Barger | Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation[END_REF].

Another population of glial cells, the astrocytes, are also involved in inflammatorytype reactions during neurodegeneration [START_REF] Liddelow | Reactive Astrocytes: Production, Function, and Therapeutic Potential[END_REF]. Like microglia, astrocytes possess the capacity to release glutamate in physiological and pathological conditions [START_REF] Vesce | Glutamate release from astrocytes in physiological conditions and in neurodegenerative disorders characterized by neuroinflammation[END_REF][START_REF] Montana | Pathological role for exocytotic glutamate release from astrocytes in hepatic encephalopathy[END_REF]. Thus, we tested whether Sa had the potential to stimulate the release of the neurotransmitter when applied to astrocyte cultures. Contrarily to our expectation, we did not observe an increase in glutamate release when astrocytes were challenged with Sa, which means that the stimulatory action of Sa on the release process was rather specific to microglial cells.

Importantly, the capacity of Sa to trigger glutamate release from microglia signifies that the excitotoxic process affecting substantia nigra DA neurons in PD may not simply result from an increased excitatory drive from subthalamic glutamatergic neurons as suggested before [START_REF] Wallace | Survival of midbrain dopaminergic cells after lesion or deep brain stimulation of the subthalamic nucleus in MPTP-treated monkeys[END_REF][START_REF] Lavaur | The noble gas xenon provides protection and trophic stimulation to midbrain dopamine neurons[END_REF] but also from a local rise of the neurotransmitter.

αSa-mediated glutamate release in microglial cells is mediated by activation of membrane receptors

The presence of αSa closely apposed to plasma membranes is coherent with the know ability of amyloid-like structures to bind to cell membrane components [START_REF] Shrivastava | Physico-Pathologic Mechanisms Involved in Neurodegeneration: Misfolded Protein-Plasma Membrane Interactions[END_REF]. This was also an indication that the stimulatory action of protein aggregates on microglial glutamate release may result from an interaction with cell membrane signal transducing receptors. Supporting this view, we found that glutamate release induced by αS was largely reduced in the presence of an antagonistic antibody for TLR2 [START_REF] Chen | A53T human α-synuclein overexpression in transgenic mice induces pervasive mitochondria macroautophagy defects preceding dopamine neuron degeneration[END_REF], a receptor subtype that is presumably involved in PD-related brain inflammation [START_REF] Watson | Regionally-specific microglial activation in young mice over-expressing human wildtype -synuclein[END_REF][START_REF] Kim | Non-cell-autonomous Neurotoxicity of αsynuclein Through Microglial Toll-like Receptor 2[END_REF]. Our result is in agreement with data reported by Gustot and colleagues (2015) showing that S amyloid fibrils can trigger inflammatory-type reactions through a TLR-2-dependent mechanism. Kim and colleagues (2013) have shown, however, that S oligomers spontaneously secreted by a neuroblastoma cell line had the potential to activate TLR-2 in microglial cells whereas fibrils produced from recombinant S were unable to do so. Such discrepancies may be due to structural heterogeneity of S oligomeric and fibrillary species in between studies [START_REF] Bousset | Structural and functional characterization of two -synuclein strains[END_REF][START_REF] Kumar | Modulation of the extent of structural heterogeneity in α-synuclein fibrils by the small molecule thioflavin T[END_REF]. Glutamate release induced by Sa was also significantly curtailed by JNJ, a synthetic antagonist for purinergic P2X7 receptors [START_REF] Letavic | Synthesis and Pharmacological Characterization of Two Novel, Brain Penetrating P2X7 Antagonists[END_REF], which is reminiscent to previous studies showing that oligomeric forms of S bind directly to this receptor subtype [START_REF] Jiang | P2X7 receptor is critical in α-synuclein-mediated microglial NADPH oxidase activation[END_REF]. The implication of P2X7 receptors in the effects of S fibrils has, however, not been reported previously.

Overall, these observations suggest that in our model system, both TLR2 and P2X7 receptors contributed to αSa-mediated glutamate release by microglial cells.

Accordingly, a combined treatment with the TLR2 antagonistic antibody and the P2X7 antagonist JNJ was required to completely suppress the stimulatory action that αSa exert on glutamate release. Still in accord with our present finding, we found that glutamate release was stimulated by selective agonists of each receptor subtype, Pam3CSK4 and BzATP and that this stimulatory effect was further enhanced by a treatment combining the two agonist compounds.

αSa-mediated glutamate release in microglial cells results from an increase in intracellular oxidative stress

Next, we aimed to better understand the nature of the mechanisms that control glutamate release in microglial cells stimulated by αSa. Experiments showing that DPI and APO, two inhibitors of the NOX superoxide producing enzyme were able to suppress glutamate release induced by αSa, suggested that ROS contributed actively to the release process. TROL, a compound that inhibits hydroxyl radical-mediated lipid peroxidation was as efficient as the NOX inhibitors to prevent glutamate release, indicating that superoxide-dependent hydroxyl radicals and/or lipid peroxyl radicals [START_REF] Franzoni | An in vitro study on the free radical scavenging capacity of ergothioneine: comparison with reduced glutathione, uric acid and trolox[END_REF]) operated as key mediators in this process. Quantitative detection of ROS with the membrane permeable fluorogenic probe CellROX confirmed that DPI, APO and TROL were all highly effective in preventing intracellular ROS production in microglia exposed to αSa. Note that glutamate release was also reported to be stimulated through NOX activation in microglial cells exposed to the bacterial inflammogen LPS [START_REF] Barger | Glutamate release from activated microglia requires the oxidative burst and lipid peroxidation[END_REF] which indicates that intracellular events contributing to glutamate release in αSa-and LPS-treated microglial cells share comonalities.

In addition to that, we found that inhibition of PI3K with LY prevented the induction of ROS by αSa which is coherent with reports showing that both TLR2 [START_REF] Lu | TLR2 ligand induces protection against cerebral ischemia/reperfusion injury via activation of phosphoinositide 3-kinase/Akt signaling[END_REF] and P2X7 ligands signal through PI3K activation [START_REF] Jiang | P2X7 receptor is critical in α-synuclein-mediated microglial NADPH oxidase activation[END_REF]. This also indicated that a PI3K-dependent mechanism controlled NOX activation in the present setting.

Coherent with this view, the stimulation of microglial cells by αSa increased the immunofluorescent signal of p47phox, a subunit of NOX2, the NOX isoform that is predominant in microglial cells [START_REF] Bedard | The NOX family of ROS-generating NADPH oxidases: physiology and pathophysiology[END_REF]2007). As expected this effect was totally reversed by PI3K inhibition, which is coherent with a previous report showing that PI3K controls NOX2 activation in microglial cells exposed to S oligomers (Jiang et al, 2015). Still consistent with our results, Hou and colleagues (2018) reported that p47phox was robustly activated by Sa in microglial cultures. However, this effect which required activation of the CD11b integrin appeared not dependent on TLR-2 signaling.

The Xc -cystine/glutamate antiporter system operates as downstream effector for Samediated glutamate release Note that we also observed that inhibition of αSa-mediated glutamate release by PI3K blockade was reproduced by Sulfa, an anti-inflammatory drug, which operates as inhibitor of the Xc -cystine/glutamate antiporter system [START_REF] Shukla | Inhibition of xc⁻ transporter-mediated cystine uptake by sulfasalazine analogs[END_REF]. This suggested that Xc -was possibly the final downstream effector for glutamate release stimulation by Sa in microglial cells. Comforting this view, the increase in glutamate release induced by αSa was accompanied by a concomitant rise in the uptake of Lcystine, a thiol aminoacid serving as precursor for the antioxidant defense tripeptide glutathione (GSH) [START_REF] Lu | Regulation of glutathione synthesis[END_REF]. Cystine uptake appeared also largely antagonized by Sulfa, confirming that the aminoacid was accumulated through the Xc -antiporter. Given that several antioxidants also prevented glutamate release induced by αSa, one may assume that the increased activity of the antiporter was closely related to the induction of oxidative stress by aggregated forms of the protein.

The neurotransmitter dopamine prevents αSa-mediated glutamate release through an antioxidant effect

The loss of DA neurons and the ensuing depletion in brain DA is another key pathological feature of PD [START_REF] Rodriguez-Oroz | Initial clinical manifestations of Parkinson's disease: features and pathophysiological mechanisms[END_REF]. Thus, we were interested in determining whether a deficit in DA could facilitate glutamate release induction by αSa.

Consistent with this view, we found that DA was potently inhibiting glutamate release in cultures that were challenged with αSa. This result is reminiscent of previous observations showing that DA can reduce some of the inflammatory responses in microglia [START_REF] Färber | Dopamine and noradrenaline control distinct functions in rodent microglial cells[END_REF] and also in bone marrow-derived macrophages [START_REF] Yan | Dopamine controls systemic inflammation through inhibition of NLRP3 inflammasome[END_REF]. The inhibitory effect of DA was entirely mimicked by a selective D1 DA receptor agonist SKF-81,297 whereas ERG, an agonist at D2 receptors failed to do so, indicating that DA effects resulted most probably from D1 receptor activation. So, the possibility that DA could reduce the immunotoxicity of S fibrils indirectly by forming complexes with them [START_REF] Béraud | Microglial activation and antioxidant responses induced by the Parkinson's disease protein α-synuclein[END_REF] seemed rather unlikely. Note that DA reduced PI3K activation and ROS production in αSa-treated microglial cultures, which signifies that DA counteracted glutamate release stimulation by inhibiting PI3K-dependent signaling.

To mimic the effects of D1 receptor activation in the present setting, we used the adenylate cyclase activator FK because this DA receptor subtype is coupled to cAMP production [START_REF] Beaulieu | The physiology, signaling, and pharmacology of dopamine receptors[END_REF]. Even if being quite effective in reducing PI3K activation, FK caused only partial inhibition of the release of glutamate induced by αSa. This suggests that both canonical and non-canonical G protein coupling of D1 receptors may occur in microglial cells. Coherent with this possibility, non-canonical G protein signaling has been reported in the immune system [START_REF] Boularan | Implications of non-canonical G-protein signaling for the immune system[END_REF].

Altogether, present data suggest that the activation of microglial cells by Sa may possibly contribute to a toxic build-up of extracellular glutamate in PD. The deficit in DA that characterizes this disorder may further aggravate this process in a vicious circle mechanism.

FIGURE 1

 1 FIGURE 1 Induction of glutamate release in microglial cells by αSa. (a) Impact of a 24hour treatment with αSa (15 and 70 µg/ml) or αSm (70µg/ml) on the release of glutamate in microglial cell cultures. Comparison with a treatment with the bacterial inflammogen LPS (10 ng/ml). Data are means ±S.E.M (n = 6) ****p<0.0001 vs controls.

FIGURE 2

 2 FIGURE 2 Glutamate release induced by αSa in microglial cells results from cell surface receptor activation. (a) Digitized images showing the presence of fibrillary aggregates in cultures of microglial cells exposed to αSa (70 µg/ml) for 24 hours. CD11b (red), S (white) and DAPI counterstained nuclei (blue). Scale bar: 10 µm. (b) Modulation of the effects of αSa (70 µg/ml) on glutamate release in microglial cell cultures treated with the MAb mTLR2 antagonist (T; 2.5 µg/ml) or the P2X7 receptor antagonist JNJ (J; 20 µM), used separately or in combination. (c) Impact that treatments with Pam3CSK4 (PAM; 1 µg/ml), a specific agonist for TLR2 and BzATP (500 µM), a specific agonist for P2X7 receptors, exert on glutamate release. Modulation of the effects of Pam3CSK4 and BzATP by MAb mTLR2 and JNJ, respectively. Impact of a combined treatment with Pam3CSK4 and BzATP on glutamate release when JNJ and MAb mTLR2, are added concurrently to the cultures. Data are means ±S.E.M (n = 8). ****p < 0.0001 vs controls.## p < 0.01, ### p < 0.001 and #### p < 0.0001 vs corresponding inflammogen.

FIGURE 3 FIGURE 4

 34 FIGURE 3 Glutamate release induced by αSa in microglial cells occurs through activation of the Xc -antiporter system. (a) Impact that Sulfa (50 µM) exerts on glutamate release in microglial cell cultures exposed to αSa (70 µg/ml). Data are means ±S.E.M (n = 6). ****p <0.0001 vs controls and #### p<0.0001 vs αSa-treated cultures. (b) Impact that Sulfa (50 µM) exerts on [ 14 C]-L-cystine uptake in microglial cell cultures exposed to αSa

FIGURE 5

 5 FIGURE 5 Dopamine prevents glutamate release induced by αSa in microglial cells through D1 receptor activation. (a) Impact of DA (10 µM) on glutamate release induced by a 24 h-treatment with αSa (70 µg/ml). Data are means ±S.E.M (n = 6). ****p<0.0001 vs controls; #### p<0.0001 vs αSa-treated cultures. (b) Impact of the D1 receptor agonist SKF (10 µM), the D2 receptor agonist ERG (10 µM), the adenylate cyclase activator FK (10 µM) and the PI3K inhibitor LY (2.5 µM) on glutamate release induced by αSa (70 µg/ml) in microglial cell cultures. Data are means ±S.E.M (n = 6-8). ****p<0.0001 vs control; ### p<0.001 and #### p<0.0001 vs αSa-treated cultures. (c) Western blot analysis of pAKT (phosphorylated at Ser473) in microglial cells exposed to αSa (70 µg/ml) for 30 min in the presence or absence of SKF-81,297 (10 µM), FK (10 µM) or LY (2.5 µM).Comparison with the expression of total AKT and GAPDH in sister cultures exposed for 24 hours to the same treatments as before. (d) Western blotting quantification of p-AKT/AKT in the same conditions as before. Data are means ±S.E.M (n = 4). *p<0.05 vs controls; ### p<0.001 and #### p<0.0001 vs αSa-treated cultures.

FIGURE 6

 6 FIGURE 6 Dopamine prevents glutamate release induced by αSa in microglial cells through an antioxidant effect. (a) Intracellular ROS production visualized with the fluorogenic probe CellROX Deep Red in microglial cells treated with αSa (70 µg/ml) for 24 hours in the presence or not of DA (10 µM), FK (10 µM) or LY (2.5 µM). Scale bar: 20 µm. (b) CellROX fluorescence intensities measured in the same conditions as in (a). Data

Figure 7

 7 Figure 7

Acknowledgments: The research leading to these results was supported by program Investissements d'Avenir [ANR-10-IAIHU-06] and the Translational Research Infrastructure for Biotherapies in Neurosciences [ANR-11-INBS-0011-NeurATRIS]. This work benefited from equipment and services from the CELIS and ICMQuant core facilities, at ICM. In particular, we wish to acknowledge expert assistance from David Akbar and Dominique Langui for inverted fluorescence microscopy and transmission electron microscopy, respectively. We wish to acknowledge helpful comments from Ana Carolina Issy, Keila Bariotto, Terence Duarte and Mariza Bortolanza.

Program Investissements d'Avenir [ANR-10-IAIHU-06] and Translational Research Infrastructure for Biotherapies in Neurosciences [ANR-11-INBS-0011-NeurATRIS].