Neurosteroid allopregnanolone regulates EAAC1-mediated glutamate uptake and triggers actin changes...

Neurosteroid AllopregnanoloneRegulates EAAC1-MediatedGlutamate Uptake and TriggersActin Changes in Schwann CellsCARLA PEREGO,1* ELIANA S. DI CAIRANO,1 MARINELLA BALLABIO,2

AND VALERIO MAGNAGHI2**1Department of Molecular Sciences Applied to Biosystems, Universita degli Studi di Milano, Milan, Italy2Department of Endocrinology, Physiopathology and Applied Biology, Universita degli Studi di Milano, Milan, Italy

Recent evidence shows that neurotransmitters (e.g., GABA, Ach, adenosine, glutamate) are active on Schwann cells, which form myelinsheaths in the peripheral nervous system under different pathophysiologic conditions. Glutamate, the most important excitatoryneurotransmitter, has been recently involved in peripheral neuropathies, thus prevention of its toxic effect is desirable to preserve theintegrity of peripheral nervous system and Schwann cells physiology. Removal of glutamate from the extracellular space is accomplished bythe high affinity glutamate transporters, so we address our studies to analyze their functional presence in Schwann cells. We firstdemonstrate that Schwann cells express the EAAC1 transporter in the plasma membrane and in intracellular vesicular compartments ofthe endocytic recycling pathways. Uptake experiments confirm its presence and functional activity in Schwann cells. Secondly, wedemonstrate that the EAAC1 activity can be modulated by exposure to the neurosteroid allopregnanolone 10 nM (a progesteronemetabolite proved to support Schwann cells). Transporter up-regulation by allopregnanolone is rapid, does not involve proteinneo-synthesis and is prevented by actin depolymerization. Allopregnanolone modulation involves GABA-A receptor and PKC activation,promotes the exocytosis of the EAAC1 transporter from intracellular stores to the Schwann cell membrane, in actin-rich cell tips, andmodifies the morphology of cell processes. Finally, we provide evidence that glutamate transporters control theallopregnanolone-mediated effects on cell proliferation. Our findings are the first to demonstrate the presence of a functional glutamateuptake system, which can be dynamically modulated by allopregnanolone in Schwann cells. Glutamate transporters may represent apotential therapeutic target to control Schwann cell physiology.J. Cell. Physiol. 227: 1740–1751, 2012. � 2011 Wiley Periodicals, Inc.

The identification of the signals regulating Schwann cell growth,development and maturation, under physiological orpathological conditions, is of great importance for theperipheral nervous system (PNS). Recently, a role for theneurosteroid allopregnanolone (ALLO) and/or theneurotransmitter GABA in such a control has been proposed(Magnaghi, 2007). ALLO is synthesized de novo in Schwann cells(Schumacher et al., 2000) and influences PNS myelinogenesisand myelin protein expression (Melcangi et al., 2005;Schumacher et al., 2007). At nanomolar concentrations, ALLOacts as a potent allosteric GABA-A receptor modulator(Majewska et al., 1986) and regulates respectively glutamatedecarboxylase (Chavez-Delgado et al., 2005) and GABAsynthesis in Schwann cells (Magnaghi et al., 2010) sustaining aGABA autocrine signaling in these cells.

Among the putative factors regulating Schwann cells it shouldbe also considered glutamate, the major excitatoryneurotransmitter and potent neurotoxin in the nervous system(Choi et al., 1987). The presence of glutamate in the Schwanncells is still an intriguing but controversial issue. Someobservations show that Schwann cells can release relevantconcentrations of the excitotoxic glutamate (Wu et al., 2005),whereas other data reveal an immunonegativity for glutamate inthe Schwann and satellite cells of the rat nodose ganglion(Schaffar et al., 1997). Schwann cells in vivo and in vitro expressa variety of ionotropic (i.e., NMDA, AMPA, or KA receptors;Fink et al., 1999; Kinkelin et al., 2000) and metabotropicglutamate receptors (Saitoh and Araki, 2010) and the enzymesinvolved in glutamatemetabolism, such asGAD (Magnaghi et al.,2010) and glutamine synthetase (GS) (Saitoh and Araki, 2010).Altogether these findings suggest a role of glutamate as asignaling molecule in the Schwann cell/neuron interactions andpredict the expression of a glutamate clearance system to

tightly control its concentration in the extracellular space. Inthe CNS, this function is accomplished by high-affinity Na-dependent glutamate transporters (excitatory amino acidtransporters, EAATs) (Danbolt, 2001). These transportersinclude glutamate-aspartate transporter (GLAST or EAAT1),glutamate transporter 1 (GLT1 or EAAT2) and excitatoryamino acid carrier 1 (EAAC1or EAAT3). High affinity glutamatetransporters have been recently identified in the PNS in vivo.GLT1 was found in the cytoplasm of Schwann cells; GLAST

Additional Supporting Information may be found in the onlineversion of this article.

Contract grant sponsor: University Research Program;Contract grant number: PUR 2007/2008.Contract grant sponsor: Association Francaise contre lesMyopathies (AFM);Contract grant number: 14163/2009.Contract grant sponsor: Compagnia di San Paolo (BandoProgramma Neuroscienze 2008-Project MOVAG);Contract grant number: 2229/2008.

*Correspondence to: Carla Perego, Department of MolecularSciences Applied to Biosystems, Universita degli Studi di Milano, ViaTrentacoste 2, 20134 Milan, Italy. E-mail: [email protected]

**Correspondence to: Valerio Magnaghi, Department ofEndocrinology, Physiolpathology, Applied Biology, Universita degliStudi di Milano, Via G. Balzaretti 9, 20133 Milan, Italy.E-mail: [email protected]

Received 1 December 2010; Accepted 10 June 2011

Published online in Wiley Online Library(wileyonlinelibrary.com), 17 June 2011.DOI: 10.1002/jcp.22898

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localized in satellite cells and in peripheral myelin, while EAAC1was detected only in the myelin layer (Carozzi et al., 2008). Inaddition, GLAST was also found on perisynaptic Schwann cells,where it may regulate the extracellular glutamateconcentration (Pinard et al., 2003). However, whether they arefunctional and/or they concur in fine tuning the equilibriumbetween Schwann cells and neurons is virtually unknown.

To shed light on the role of glutamate in Schwann cells ourstudies aimed at characterizing the glutamate transportersystems. As glutamate may be substrate for GABA synthesis,and glutamate transporters may be involved in this process(Bak et al., 2006) we also verified whether they were functionaland regulated by ALLO. Finally, we identified the molecularmechanism by which ALLO modified glutamate transportersactivity in Schwann cells.

Materials and MethodsAnimals

For the Schwann cell cultures, the minimal number (about30 animals) of Sprague–Dawley 3-day-old rats (Charles River,Calco, Italy) was used. For other studies, adult (3-month-old) ratswere anesthetized with an intraperitonal injection of pentobarbital(30mg/kg), to minimize pain, immediately killed, and explantedtissues (brain, adipose tissue, sciatic nerve) frozen or fixed untilanalysis. All experiments were performed according toinstitutional guidelines of the Ethical Committee of our Universityand in compliancewith the policy on the use of animals approved bythe European Communities Council Directive (86/609/EEC).

Schwann cell cultures

Schwann cell cultures were obtained by the method of Brockes(Brockes et al., 1979) with minor modifications (Magnaghi et al.,2006). Sciatic nerves from 3-day-old rats were digested with 1%collagenase and 0.25% trypsin (Sigma, Milan, Italy) then filteredthrough a 30mm nylon membrane and centrifuged 10min at 280g.Pellets were solubilized in Dulbecco’s modified Eagle’s medium(DMEM) (Serotec, Oxford, UK) supplemented with 10% foetal calfserum (FCS; Invitrogen, San Giuliano Milanese, Italy) and platedonto 35mm Petri dishes. Twenty-four hours after plating, themedium was supplemented with 10mM arabinoside C (Sigma)then after 48 h the cultures were treated with a stream of coldDMEM-FCS 10%. The remaining cells were plated in DMEM-FCS10% supplemented with 10mM forskolin (Sigma) and bovinepituitary extract 200mg/ml (Invitrogen). Cells became confluent in10 days. Immunopanning for final purification was carried outincubating the cells for 30min with mouse anti rat Thy1.1 antibody(Serotec, Oxford, UK Italy) followed by 500ml of baby rabbitcomplement (Cedarlane, Ontario, Canada). The cell suspension(3� 105) was seeded on 6 cmPetri dishes and grown in presence of2mM forskolin and bovine pituitary extract 200mg/ml. At the thirdpassage in vitro Schwann cell cultures were differentiated bytreatment for 48 h with 4mM forskolin, then maintained inserum-free medium 24 h before experiments.

Pharmacological treatments

Schwann cell cultures were treated for the indicated time with10 nM or 1mM ALLO or isoALLO (isopregnanolone, 3alpha-hydroxy-5alpha-pregnan-20-one) before processing for uptake orimmunofluorescence experiments. To study the mechanisms ofallosteric ALLO modulation, the cells were incubated for 30minwith the following drugs: 100mM bicuculline, 1mM muscimol,100mM D-2-amino-5-phosphonovaleric acid (AP5) and 100mM(EDTA) in the presence or the absence of 10 nMALLO. To identifythe intracellular pathways, the cells were treated for 30min with10mM bisindolylmaleimide I, or 1mM 12-O-tetradecanoylphorbol-13-acetate (TPA) with or without ALLO 10 nM. To investigate the

role of protein biosynthesis and actin dynamics, the cells werepreincubated for 1 h with 10mg/ml cycloheximide or for10min with 2mM cytochalasin D, respectively, before ALLOincubation. To analyze the mechanism of ALLO-mediated EAAC1re-localization, the cells were treated with 20mMmonensin in theabsence or presence of 10 nM ALLO or preincubated for 1 h with80mMdynasore before ALLO incubation. For the cell proliferationassay, the cells were treated with10 nM ALLO, 10mM HIP-A,10mM DL-threo-b-benzyloxyaspartic acid (TBOA; Tocris, Bristol,UK). All drugs, except TBOA, were obtained by Sigma.

Reverse transcription-polymerase chain reaction (RT-PCR)

Total RNA was isolated by phenol–chloroform extraction(Chomczynski and Sacchi, 1987); 1mg of total RNA from eachsample was treated for 15min with deoxyribonuclease I(Invitrogen), and then reverse transcribed at 378C for 60min using200U of Moloney Murine Leukemia Virus-reverse transcriptase(Invitrogen) in the presence of random hexamer primers(Invitrogen). RT efficiency and data normalization were measuredby amplification of 18s rRNA rat transcript (accession numberM11188). PCR amplification was carried out using specific primers,designed with the assistance of the Primer3 software (http://primer3.sourceforge.net/) in the 30 end of each cDNAs. Thefollowing primers were used: forward50-AAGTATCACAGCCACAGC-30 (1490–1507), reverse50-TCTCTGGTTCATTGTCCTGG-30 (1803–1784) for GLAST(Gene ID 29483); forward 50-GCTGCTGGATAGAATGAGAACTTCGG-30 (1524–1549),reverse 50-TTCCAAGGTTCTTCCTCAACACTGC-30(1820–1796) for GLT1 (Gene ID 29482) forward 50-TTTGCCTTGGAGCCCACAATCC-30 (1629–1651), reverse50-TTTCCCTTCGGTTCATCCCACC-30 (1960–1939) forEAAC1 (Gene ID25550). The amplification reactions were carriedout for 40 or 42 cycles (annealing for 30 sec at 588C) in a finalvolume of 25ml containing 20mM Tris–HCl, pH 8.3, 50mM KCl,1.5mM MgCl2, deoxynucleotides (200mM each), 1U of EuroTaqDNApolymerase (Euroclone, Pero, Italy), 1mMof each primer and2 or 6ml of cDNA as template. A final elongation at 728C for 10minwas performed. Additional samples containing the sameRNA, usedas negative controls, were treated following the same amplificationconditions except that the reverse transcriptase was omitted. Thepredicted amplified products of EAATs of 310 bpwere analyzed on2% agarose gels and photographed.

SDS–PAGE and Western blotting analyses

Brain (positive control), adipose tissue (negative control) and1.5� 106 differentiated Schwann cells were lysed in 40ml lysisbuffer (150mM NaCl, 50mM Tris–HCl, 1mM EDTA, 1% TritonX-100, 1mM phenylmethylsulfonylfluoride, and 1mg/ml aprotinin,peptin, and leupeptin). After 30min at 48C, lysates werecentrifuged at 13,000 rpm for 15min, and 10mg (P2) or 30mg oftotal extracted proteins were denatured in SDS sample buffer at958C for 5min, and underwent 9% SDS–PAGE as described(Perego et al., 2000a). They were then transferred ontonitrocellulose (Schleicher and Schuell, Dassel, Germany). P2extract from rat brain was obtained as described (Perego et al.,2000b). The blots were probed with rabbit anti-EAAC1,anti-GLT1, anti-GLAST (Alpha Diagnostic, San Antonio, TX) oranti-actin (Sigma) primary antibodies, followed by incubation withperoxidase-conjugated anti-rabbit IgG as secondary reagents, andvisualized by ECL (Perkin-Elmer Life Science, Boston, MA). TheX-ray films were analyzed by densitometry and the resultsquantified using NIH Image 1.59 software.

Immunofluorescence analysis

For assessment of cell purity and other immunofluorescenceanalysis, cultures were fixed in 4% paraformaldehyde (PFA) or ice

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cold methanol, permeabilized with 0.5% Triton X-100 (TX-100)and immunostained as described (Perego et al., 1999). TheSchwann purity (more than 98%) was tested with a specificantibody against the Schwann cell marker glycoprotein P0, asalready used for other studies (Magnaghi et al., 2004, 2010). Cellsimmunopositive for P0 were counted under an Axioskop200microscope (Zeiss, Berlin, Germany). For immunofluorescenceanalyses the following primary antibodies were used: polyclonalanti-EAAC1, anti-GLT1 and anti-GLAST (Alpha Diagnostic),anti-myelin basic protein (MBP, Chemicon, Temecula, CA),anti-early endosomes antigen-1 (EEA1, Sigma), anti-Rab11 (Sigma).The actin cytoskeleton was stained with rhodamine-conjugatedphalloidin (Sigma). To label the endocytic pathway, the cells wereincubated on ice with concanavalinA biotin-XX conjugate (ConA,Molecular Probes/Invitrogen) for 30min, and then cultured for 1 hat 378C before fixation and staining as previously described(D’Amico et al., 2010). The secondary antibodies (fluorescein- orrodhamine-conjugated anti-rabbit and fluorescein-conjugatedanti-rat IgG antibodies) came from Jackson Immunoresearch(West Grove, PA).

Coronal sections (12mm) of rat sciatic nerve (mounted onpoly-L-lysine-coated slides) were fixed in PFA 4% and incubated30min at room temperature in 1:500 FluoromyelinTM redfluorescent myelin stain (Molecular Probes). Slides were thenincubated overnight at 48C in phosphate buffer containing 0.25%bovine serum albumin (Sigma), 0.1% Triton X-100 and the primaryrabbit anti-EAAC1. The following day slides were washed twotimes and incubated 2 h at room temperature with the specific goatanti-rabbit Alexa-FITC-488 (Molecular Probes, San GiulianoMilanese, Italy; diluted 1:800) secondary antibody. After washingslides were mounted using VectashieldTM (Vector Laboratories,Burlingame, CA) and nuclei stained with 40,6-diamidino-2-phenylindole (DAPI; Sigma).

Controls for antibodies specificity included a lack of primaryantibodies. Cells and sciatic nerves were analyzed by fluorescencemicroscopy (Axiovert; Zeiss) and the image analysiswas done usingAdobe Photoshop 5.0.2.

Cell transfection

About 5� 104 Schwann cells were seededonto glass coverslips anddifferentiated as above described. Twenty-four hours afterdifferentiation, they were transfected with 1mg of pEGFP-rabbitEAAC1 by means of lipofection (LipofectamineTM 2000 reagent,Invitrogen) (D’Amico et al., 2010) and 24 or 48 h after transfection,the cells were processed for TIRF microscopy.

Total internal reflection fluorescence microscopy (TIRFM)

Twenty-four hours after transfection, differentiated Schwann cellsplated onto glass coverslips were fixed and imaged through a TIRFmicroscope (Axiovert; Zeiss) equippedwith anArgon laser at 378Cusing a 100� 1.45 numerical aperture (NA) oil immersionobjective. Green fluorescence was excited using the 488 nm laserline and imaged through a band-pass filter (Zeiss) onto a Retiga SRVCCD camera (D’Amico et al., 2010). For the time-lapseexperiments, single-cell imaging under TIRF illumination wascarried out at 3 frames/min for 7min before and after incubation at378C with 10 nM ALLO. In experiments with dynasore, cells werepre-treated in drug-containing solution (80mM for 1 h) before theexperiment. In experiments with monensin, cells were incubatedwith 20mM monensin alone or together with 10 nM ALLO. Up tofive cells were imaged on each coverslip, in three independentexperiments.

Image processing. For each recorded cell image, eight 50� 50-pixel regions were randomly selected within the cell, and the averagefluorescence intensity and its associated standard deviation in eachframe were calculated using the Image-ProPlus software (MediaCybernetics, Bethesda, MD). The fluorescence intensities in thevarious frames were normalized to their respective initial averageintensities (F0¼ 100%) and plotted against time. Fluorescence

intensities were corrected for bleaching during the acquisition of serialimages (bleaching was estimated by imaging paraformaldehyde-fixedcells).

[3H] D-Aspartic acid uptake

About 2� 104 Schwann cells/well were seeded on 24-well plates,and differentiated as above reported before uptake experiments.They were first washed twice in sodium-free solution (150mMcholine chloride, 4mM KCl, 1mM CaCl2, 1mM MgCl2, 5mMHEPES, pH 7.5) and then assessed for [3H]D-aspartate uptake asdescribed (Perego et al., 1997). Single-shot [3H]-D-aspartate(3mCi/ml; specific activity 37Ci/mmol, Amersham Biosciences,Milano, Italy) uptake was performed in 150ml of Na-dependent(150mMNaCl, 4mMKCl, 1mMCaCl2, 1mMMgCl2, 5mMHEPES,pH 7.5) orNaþ-independent (150mMCholineCl, 4mMKCl, 1mMCaCl2, 1mM MgCl2, 5mM HEPES, pH 7.5) uptake solution for10min at room temperature. Amino acid uptake was stopped bywashing the cells twice in ice-cold sodium-free solution. The cellswere dissolved in 150ml of SDS 1% for liquid scintillation counting.For transport inhibition experiments, HIP-A (a kind gift of Prof. DeMicheli C. and Prof.ssa Conti P.; Funicello et al., 2004) or DHK(Sigma) was added to the uptake solution at the indicatedconcentrations. In ALLO experiments Schwann cells wereincubated with 10 nM ALLO or vehicle (control) for 30min beforeuptake assays. The Na-dependent [3H]D-aspartate (measured inNaCl solution) was calculated without subtraction of the uptake inthe presence of CholineCl (ChCl).

Cell proliferation assay

Schwann cells (2� 104) were seeded on 24 well plates and treatedwith the indicated drugs in the presence of 100mM 5-bromo-20-deoxyuridine (BrdU; Sigma) to assess their proliferation index.Twenty-four hours after treatment the cell cultures were washedin TBS buffer (500mM NaCl and 20mM Tris–HCl pH 7.4) thenfixed for 20min in PBS-PFA 4% and denatured 30min at 378CwithHCl 2N. After washing in TBS the cultures were incubatedovernight at 48C with mouse monoclonal anti-BrdU (Sigma),diluted 1:1000, in phosphate buffer plus 0.25% bovine serumalbumin (Sigma), 0.1% Triton X-100. The following day, slides werewashed and incubated 2 h with goat anti-mouse Alexa-FITC-488(Molecular Probes; diluted 1:1,000) secondary antibody and nucleiwere stained with DAPI. The cells were analyzed by fluorescencemicroscopy (Axiovert; Zeiss) and counted using Image-Pro Plus 6.0(Media Cybernetics).

Data analysis

Data were statistically evaluated by GraphPad Prism 4.00(San Diego, CA). Statistical significance between groups wasdetermined by means of an unpaired Student’s t-test. P values of<0.05 were considered significant.

ResultsHigh-affinity glutamate transporters expression inSchwann cells

Wefirst verified the presence of themRNAs coding for the highaffinity glutamate transporters in Schwann cells in vitro.RT-PCR analysis performed with specific primers for thedifferent glutamate transporter subtypes revealed the presenceof EAAC1 mRNA in Schwann cells (Fig. 1A). Neither GLASTnor GLT1 transcripts were detected in Schwann cells, even athigher amplification cycles (42 cycles) or using higher quantityof template (6ml) as substrate (Fig. 1A).

The Western blot analysis performed with specificanti-glutamate transporter antibody confirmed EAAC1expression in Schwann cells. As shown in Figure 1B, bands of 66and 120 kDa corresponding to the reported molecular weightsof monomeric and oligomeric EAAC1, respectively were


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detected in Schwann cells and P2 brain lysates (positivecontrol). Although the reported antibodies have beenpreviously used (Gonzalez et al., 2007; D’Amico et al., 2010), tofurther confirm the antibody specificity in our model, anadipose tissue lysate was analyzed as a negative control and nobands were detected. In agreement with the RT-PCR findings,

we did not detect the expression of either GLAST and GLT1(Fig. 1B).

Double immunofluorescence analyses with antibodiesagainst EAAC1 transporter and myelin basic protein (MBP), aselective marker of Schwann cells, revealed the presence ofEAAC1 on the cell surface of Schwann cells, a localizationconsistent with its functional role (Fig. 2A). Interestingly, thetransporter labeling was particularly evident in actin-rich

Fig. 1. Expression of high affinity glutamate transporters inSchwann cells. A: RT-PCR analysis reveals the presence of EAAC1mRNA transcripts. Total RNAwas extracted fromSchwann cells andRT-PCR was carried out with primers specific for the differentglutamate transporter types. The transcripts of 310 bp (for EAAC1)and 251 bp (for 18s) were detected in the Schwann cells. Total RNAfromratbrainwasusedas apositive control.No transcript forGLASTand GLT1 were observed in Schwann cells; (T) RNA with reversetranscriptase; (NT)RNAwithout reverse transcriptase; (Mk)marker.B: Western blot analysis reveals the expression of EAAC1transporter. Ten micrograms of brain P2 fraction, 30mg of adiposetissue (Ad) and 30mg of Schwann cells (Sc)whole lysate extracts wereseparated onto a 9% SDS–PAGE and the expression of transporterswas determined using isotype specific anti-glutamate transporterantibodies. P2 lysate was used as a positive control while adiposetissue was used as negative control. EAAC1 was detected only in theP2 and Schwann cell lysates.

Fig. 2. Immunolocalization of EAAC1 in Schwann cells. A: EAAC1localizes to the plasma membrane and in intracellular vesicularstructures of Schwann cells. Cells were fixed in methanol and doublestainedwith anti-MBP (red), amarkerof differentiatedSchwann cells,and anti-EAAC1 (green) antibodies. Bar: 10mm. The EAAC1localization in punctuate structures is evident in the particular shownat higher magnification (3T) in the EAAC1 panel. B: EAAC1accumulates in Schwann cell tips, positive for actin. Cellswere fixed inmethanol and double stainedwithTRITC-conjugated phalloidin (red)and anti-EAAC1 antibody (green). Bar: 5mm. Arrowheads indicatethe localization of EAAC1 in vesicular structures. C: EAAC1 (green)colocalizes with markers of the endocytic/recycling pathway (red).Schwann cells were double stained with EAAC1 (green) andinternalized texas red-conjugated concanavalin A (marker of theendocytic pathway), EEA1 (marker of early endosomes) or Rab11(markerof the recycling compartment) (red), fixedandanalyzed.Theyellow/orange staining demonstrates the colocalization of EAAC1with markers. Nuclei are shown in blue (DAPI). Bar: 10mm. Aparticular at higher magnification is shown. Bar: 5mm. D: EAAC1localizes in Schwann cells in vivo. EAAC1 immunopositivity (in green)was shown in some Schwann cells (white arrow) surrounding themyelin fibers (in red) as shown in coronal sections (12mm) of ratsciatic nerve. Nuclei were stained in blue with DAPI (arrowheads).Bar: 20mm.



Schwann cells tips and lamellipodia (Fig. 2B). In addition, EAAC1staining was also detected in bright intracellular vesicularstructures (Fig. 2A, inset) that largely colocalized withinternalized concanavalin-labeled glycoproteins (Fig. 2C).Double immunofluorescence experiments revealed somecolocalization of EAAC1 with EEA1 (a marker of earlyendosomal compartments) in peripheral structures and withRab11 (the marker of the endocytic compartment) in theperinuclear region (Fig. 2C). A similar localization of EAAC1 inthe plasma membrane and in endocytic/recyclingcompartments has been reported in epithelial and neuronalcells (Gonzalez et al., 2007; D’Amico et al., 2010).

The EAAC1 localization was tested also in vivo. In thecoronal section of rat sciatic nerve, some Schwann cells (ingreen) surrounding the myelinated fibers (in red) wereimmunopositive for EAAC1 confirming that Schwann cells invitro aswell in vivo express this glutamate transporter (Fig. 2D).

Uptake experiments with [3H]D-aspartate (a non-metabolized substrate; Danbolt, 2001) revealed the existenceof a minor Na-independent and a prevalent Na-dependenttransport activity, suggesting the presence of functional highaffinity glutamate uptake systems in Schwann cells (Fig. 3).Subtype-specific expression of glutamate transporters wascharacterized pharmacologically using 3-hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-4-carboxylic acid(HIP-A), a non selective high affinity glutamate transporters’blocker (Funicello et al., 2004) and dihydrokainate (DHK) aselective GLT1 inhibitor (Arriza et al., 1994). The Naþ-dependent uptake of [3H]D-aspartate was completely inhibitedby 10mM HIP-A but not by 0.1mM DHK, suggesting that theuptake of glutamate in these cells is not driven by GLT1 andconfirming the RT-PCR andWestern blot experiments (Fig. 1).

ALLO regulates [3H]D-aspartate uptake in Schwann cells

Having established the functional expression of glutamatetransporters we then investigated whether they are modulatedby ALLO. Schwann cells were incubated with 10 nM or 1mMALLO or its inactive stereoisomer (isoALLO) (Lambert et al.,1995), for 30min and the [3H]D-aspartate transport wasmeasured in the presence of sodium. As shown in

Figure 4A both the 10 nM and 1mMALLO concentrationswereable to significantly increase the [3H]D-aspartate accumulation.No significant changes in the [3H]D-aspartate uptake wereobserved in the presence of isoALLO, confirming the selectivityof the neurosteroid effect. ALLO induced [3H]D-aspartatetransport up-regulation was time-dependent with a peak effectaround 30–40min; the effect was transient and disappearedafter 120min of stimulation (Fig. 4B). No change in theNa-independent [3H]D-aspartate uptake were measured in thesame experimental conditions (data not shown).

Mechanisms and effects of glutamate transportup-regulation induced by ALLO in Schwann cells

ALLO was active at nanomolar concentrations, suggesting thatthe effect might be mediated by an allosteric modulation ofGABA-A receptor. We tested this hypothesispharmacologically, by co-incubating Schwann cells with ALLOand bicuculline (a selectiveGABA-A antagonist). The treatment

Fig. 3. A Na-dependent [3H]D-aspartate transport activity ispresent in Schwann cells. [3H]D-aspartate uptake was measured inSchwann cells in the presence of NaCl (Na-dependent) or ChCl (Na-independent), as indicated. TheNa-dependent transport activity wascompletely inhibited by 10mM HIP-A, a non-selective, high-affinityglutamate transporter inhibitor. No significant changes weredetected in the presence of 0.1mM DHK, a GLT1-specific inhibitor.Data are expressed as cpm/well/15min and represent themeanWSEMof at least three independent experiments performed intriplicate (MMP<0.01 vs. Ctr).

Fig. 4. Modulation of [3H]D-aspartate uptake in Schwann cells byALLO. A: ALLO induces a specific [3H]D-aspartate upregulation inSchwann cells. Cells were incubated with 10 nM and 1mM ALLO orisoALLO (a non active ALLO stereoisomer) for 30min and the[3H]D-aspartate uptake was measured in the presence of 150mMNaCl. Data are expressed as cpm/well/15min and represent themeanWSEMof at least three independent experiments performed intriplicate (MMP<0.01 vs. Ctr). B: ALLO induces a time-dependent[3H]D-aspartate upregulation. Cells were incubated with 10nMALLO for the indicated times and the transporter activity wasmeasured by means of [3H]D-aspartate uptake in the presence ofNaCl. Transport up-regulation is transient and reaches a pick after30–40min. Data are expressed as fold increase over control, andrepresent themeanWSEMof at least three independent experimentsperformed in triplicate (MP<0.05; MMP<0.01, vs. 0min).


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with 100mM bicuculline did not significantly affect aspartateuptake in control conditions but it completely prevented theALLO-induced uptake up-regulation (Fig. 5A). To confirm theGABA-A involvement, we measured the [3H]D-aspartate

uptake in the presence of 1mMmuscimol, a selective GABA-Areceptor agonist. Muscimol induced a significant (P< 0.05)increase in aspartate uptake, and the effect was quantitativelysimilar to that observed in ALLO treated samples; co-incubation with 10 nM ALLO induced an additive effect versusmuscimol alone that was not statistically significant (Fig. 5A).

Although ALLO has also been demonstrated to activateNMDA receptors or Ca2þ-currents (Keller et al., 2004;Sedlacek et al., 2008), neither the selective NMDA antagonistAP5 (50mM), nor the extracellular calcium chelator EDTA(100mM) prevented the stimulatory effect of the neurosteroidon aspartate uptake in our experimental system (Fig. 5B).

We then investigated the intracellular pathway downstreamto ALLO-mediated allosteric activation of the GABA-Areceptor. As glutamate transporter activity and expressionhave been clearly shown to be modulated by protein kinase C(PKC) (Gonzalez and Robinson, 2004b) we evaluated theinvolvement of this pathway in the ALLO action. As shown inFigure 5C, the treatment with the selective PKC inhibitorbisindolylmaleimide (10mM) prevented the ALLO-induced[3H]D-aspartate upregulation. The PKC involvement wasfurther confirmed by treatment with its activator, the phorbolester TPA. One micromolar TPA alone induced a [3H]D-aspartate transport up-regulation identical to that observedafter ALLO treatment, but no cumulative effect was detected inthe presence of ALLO (Fig. 5C).

Finally, we investigated the molecular mechanism by whichALLO induced the glutamate transport up-regulation. Firstly,we excluded the possibility that protein biosynthesis may beinvolved in ALLO-induced [3H]D-aspartate uptake. As shown inFigure 6A theALLO-mediated effect on the transporter activitywas not prevented by incubation with 10mg/ml cycloheximide(protein biosynthesis blocker) treatment (Fig. 6A). Inagreement with this finding, we did not detect a significantchange in the expression of EAAC1 transporter in Schwanncells treated for 1 or 2 h with ALLO, with respect to controlsamples (Fig. 6B). Conversely, we found that the treatmentwith2mM cytochalasin D (an actin depolymerizing drug) completelycounteracted the ALLO effect (Fig. 6A), thus indicating theinvolvement of a cytoskeleton rearrangement in thephenomenon. Coherently, we found that ALLO incubation for30min determined a dynamic rearrangement of the corticalactin cytoskeleton (Fig. 7A); actin accumulated at the leadingedge of cells and in filopodia, whose number and length wassignificantly increased by the treatment. A parallel translocationof the transporter from the intracellular stores to the plasmamembrane, in actin-rich structures, was detected (Fig. 7B).

To confirm this possibility, we transfected the cells with aEGFP-EAAC1 construct (D’Amico et al., 2010) and monitoredits localization by TIRFM, which allows the selective excitationof labeled transporters located in or immediately below theplasma membrane (100 nm above the glass coverslip),contemporarily excluding the signals arising from structures inthe cell interior (Axelrod, 2001; Fig. 8A). In basal conditions, theEGFP-EAAC1 construct prevalently localized in intracellularvesicular structures similar to those observed with theendogenous EAAC1, thus excluding the possibility that theywere artifactually induced by the construct overexpression.Staining was also detected at the plasma membrane. AfterALLO incubation, the surface density of EGFP–EAAC1increased and the transporter was tethered to actin-positiveSchwann cell tips, whose presence was increased by ALLOtreatment (Fig. 8A). This effect was reversed by the treatmentwith 2mM cytochalasin D (Fig. 8A) in agreement with acytoskeleton rearrangement in the ALLO-inducedupregulation of EAAC1.

A quantitative analysis support this conclusion (Fig. 8B).Time-lapse TIRF imaging was used to measure the surfacerecruitment of EAAC1 transporters under ALLO stimulation. If

Fig. 5. Mechanisms of ALLOs induced aspartate transportupregulation. A: The ALLO effect is mediated through GABA-Areceptor activation. Schwann cells were incubated for 30min with100mMbicuculline (selectiveGABA-Aantagonist) or 1mMmuscimol(selective GABA-A agonist) in the absence (CTR) or presence(ALLO)of 10nMALLO.The [3H]D-aspartateuptakewasmeasured inthe presence of NaCl. Data are expressed as cpm/well/15min andrepresent themeanWSEMof at least three independent experimentsperformed in triplicate (MMP<0.01; MP<0.05 vs. CTR). B: The ALLOeffect on [3H]D-aspartate uptake is not mediated via NMDAreceptors or via Ca2R-currents. Schwann cells were incubated for30min with 100mM AP5 or 100mM EDTA in absence (CTR) orpresence (ALLO) of 10 nM ALLO and the Na-dependent [3H]D-aspartate uptake was measured. Data are expressed as fold increaseover relative controls and represent themeanWSEMof at least threeindependent experiments performed in triplicate (MP<0.05;MMP<0.01 vs. CTR). C: PKC activation mediates the ALLO-induced[3H]D-aspartate uptake. Schwann cells were incubated for 30minwith 10mMbisindolylmaleimide or 1mMTPA in the absence (CTR) orpresence (ALLO) of 10 nM ALLO before [3H]D-aspartate uptakemeasurements in NaCl solution. Data are expressed as cpm/well/15min and represent the meanWSEM of at least three independentexperiments performed in triplicate (MMP<0.01 vs. CTR).



the vesicles containing the EGFP-transporter move from thecytosol to the plasma membrane within the evanescent field,the fluorescence signal recorded by TIRFM shouldprogressively increase. Figure 8B shows a representative imagesequence together with the averaged fluorescence intensitycurves. Visual inspection of the sequence indicated thepresence of EGFP–EAAC1 in a vesicular structure at time zero(arrow), the punctuate staining progressively disappeared andthe fluorescence signal became more uniformly distributed tothe plasma membrane. Note also the progressive recruitmentof EGFP–EAAC1 in cell tips (arrowhead). In agreementwith thisobservation, the total fluorescence intensity measured duringthe 6-min recording significantly increased in the presence ofALLO (1.14� 0.04, P< 0.001). Interestingly, ALLO treatmentmodified the morphology of cell processes (Fig. 8C) whichappeared broadened and enlarged. This phenomenon isparticularly evident in the recorded video [see SupplementaryMaterial 1 (control) and Supplementary Material 2 (ALLO)].

Increased EAAC1 surface expression after ALLO treatmentmay be the result of increased transporter delivery fromendocytic compartments to plasma membrane, decreasedinternalization or a combination of the two processes. Tocharacterize the molecular mechanism of ALLO-inducedEAAC1 upregulation we used an inhibitor of endosomalrecycling, the carboxylic ionophoremonensin (Basu et al., 1981;Sorkina et al., 2005) and an endocytosis blocker, the cell-permeant dynamin inhibitor dynasore (Thompson andMcNiven 2006; see Fig. 9). A 30min incubation with 20mMmonensin decreased the basal [3H]D-aspartate uptake, andcompletely prevented the ALLO-mediated effect (Fig. 9A). As

the ionophoremonensin, by changing the ion gradient, can havea direct inhibitory effect on EAAC1 function,we tested the drugeffect on EAAC1 surface expression by TIRFM. Monensinincubation (20mM, for 6min) did not change the EGFP–EAAC1surface expression but completely prevented the increase insurface fluorescence induced by co-application of 10 nM ALLO(Fig. 9B). Conversely, dynasore incubation (80mM, 1 h, 378C)had no effect on the [3H]D-aspartate uptake under restingconditions and did not prevent the ALLO-induced EAAC1upregulation (Fig. 9C). Similar results were obtained by TIRFManalysis (Fig. 9D), in which dynasore preincubation slightlyincreased the EGFP–EAAC1 surface expression but did notprevent the ALLO-induced increase in surface fluorescenceintensity during the 6-min recording (P< 0.001). Takentogether, these data indicate that ALLO up-regulates glutamatetransporter uptake in part by increasing its delivery to Schwanncell surface.

Finally, to correlate the effect of ALLO with the biologicalfunction of EAAC1 in Schwann cells, we tested whether ALLOchanges Schwann cell proliferation in presence of glutamatetransporter inhibitors, such as HIP-A and/or TBOA (Fig. 10).We found that 24 h after treatment, ALLO induced a significantincrease in cell proliferation that was reversed by incubationwith the non-selective glutamate transporter inhibitor HIP-A(Fig. 10). Similarly, incubation with TBOA, a potent glutamatetransporter inhibitor (Shimamoto et al., 1998), prevented theALLO effect and induced a significant decrease of Schwann cellproliferation (Fig. 10). These data suggest that glutamatetransporters mediate the positive effect of ALLO on Schwanncell proliferation.

Fig. 6. ALLO effect on glutamate transporter expression. A: The ALLO effect does not require protein neosynthesis but an intact actincytoskeleton. Schwann cellswere preincubatedwithCycloheximide (10mg/ml for 1 h) orCytochalasinD (2mMfor 15min), and then treatedwith10 nMALLO for 30min, before [3H]D-aspartate uptakemeasurements in the presence ofNaCl. Data are expressed as fold increase over relativecontrolandrepresentthemeanWSEMoftwoindependentexperimentsperformedintriplicate(MP<0.05vs.CTR).B:ALLOeffectdoesnotchangethe total expression of GLAST and EAAC1. Schwann cells were incubated with 10 nM ALLO for 60 or 120min. Then cells were lysed and theexpression of EAAC1 in whole lysates was determined byWestern blotting experiments. Actin was stained in the same blot to control proteinloading.Arepresentative immunoblot isshown.C:QuantitativeanalysisofglutamatetransporterexpressionafterALLOtreatments.Bandswerequantified by densitometry. Datawere normalized for actin expression and are presented as fold increase over control. Mean valuesWSDof twoindependent experiments.


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Discussion

Herewe report for the first time a complete characterization ofthe glutamate transporters system in the Schwann cells,providing evidences that: (1) Schwann cells express EAAC1 atthe plasma membrane and in the intracellular vesicularcompartments involved in the endocytic/recycling pathways;(2) the progesterone’s metabolite ALLO up-regulatesglutamate transporter activity in Schwann cells by allostericmodulation of GABA-type A (GABA-A) receptor; (3) ALLOmodulation does not involve protein’s neo-synthesis, while it ismediated by PKC-dependent mechanisms and causes the actincytoskeleton reorganization and the redistribution oftransporters from intracellular stores to the cell surface.

The identification of the glutamate transporters system inthe PNS has been recently performed in vivo, providingobservations that are in accordance with our results. In the ratsciatic nerve, EAAC1 and GLAST preferentially stained themyelin layer, although GLT1 was found in the cytoplasm ofSchwann cells (Carozzi et al., 2008). Similarly, we found EAAC1staining in the Schwann cell membranes and in intracellularvesicular structures of Schwann cells, a localization alreadydescribed in neuronal and glioma cells (Gonzalez et al., 2007).On the contrary, we could not detect GLAST and GLT1expression by Western blotting and uptake experiments. Thisdiscrepancy may probably rely on our in vitro system, which iscompletely devoid of neuronal cells. Indeed, it is known thatneuronal factors support the expression of the GLAST andGLT1 glutamate transporters in the CNS (Swanson et al., 1997;Perego et al., 2000a).

Although EAAC1 is predominantly located in neurons in theCNS, its presence in Schwann cells is not surprising. Indeed,EAAC1 is not an exclusive neuronal transporter: it has beencloned from intestine (Kanai and Hediger, 1992) and alsodetected in non-excitable cells such as epithelial and prostatecells (Franklin et al., 2006; D’Amico et al., 2010). EAAC1 hasbeen involved in multiple processes, distinct from glutamateclearance, that might be relevant for Schwann cell physiologysuch as glutathione metabolism (Aoyama et al., 2006) and/orGABA synthesis (Bak et al., 2006).

Our data indicate that this transporter is functional inSchwann cells and its activity can be rapidly and transientlyupregulated by nanomolar concentrations of ALLO. The ALLOeffect on transport upregulation is mediated via GABA-Areceptor, as shown by the fact that it was completely preventedby bicuculline (GABA-A antagonist) co-incubation andmimicked by incubation with muscimol (GABA-A receptoragonist). ALLO is a neurosteroid, potentmodulator of all majorGABA-A receptor isoforms (Majewska et al., 1986; Belelli andLambert, 2005) with the peculiarity that at low nanomolarconcentrations it enhances GABA currents by actingallosterically on the GABA-A receptor, while in asubmicromolar-to-micromolar range it directly activates theGABA-A receptor channel complex (Callachan et al., 1987). Inthis context, it must be highlighted that Schwann cells express afunctional GABA-A receptor which is allosterically activated byneurosteroids such as ALLO (Magnaghi, 2007).

We studied the intracellular pathway whereby ALLOenhances [3H]D-aspartate uptake andwe found that PKC is alsoinvolved in such a mechanism. Indeed, the PKC inhibitorbisindoleylmaleimide decreased the basal activity of the

Fig. 7. ALLO effect on glutamate transporter localization. A: ALLO incubation modifies the actin cytoskeleton. After incubation with 10 nMALLO for 30min, Schwann cells were fixed, stainedwith phalloidin (actin) and imaged by epifluorescence. Arrows indicate actin redistribution atSchwann cells edges. Bar: 10mM.B:ALLOcauses the redistribution of EAAC1.After incubationwith 10 nMALLO for 30min, Schwann cellswerefixed, double stainedwith phalloidin andEAAC1and imagedby epifluorescence.Arrowheads indicate the enrichment of EAAC1 in actin-positivetips. Bar: 5mM.



transporter and prevented the ALLO stimulatory effect.Moreover TPA (a PKC activator) did not act additively withALLO to increase uptake activity, suggesting that these twoagents act via the same pathway. It must be emphasized thatamong the signaling linked to GABA-A receptor modulation,PKC activation may represent a possible mechanism (Krisheket al., 1994; Fancsik et al., 2000).

The rapid action of ALLO, the involvement of the PKCpathways and the localization of transporters in endocytic/recycling vesicles in basal conditions support the possibility thatuptake upregulation could be the result of transporters’redistribution from intracellular stores to the plasmamembrane. In line with this possibility, our data ofimmunofluorescence and TIRFM analysis showed increaseddensity of EAAC1 transporters at the plasma membrane. Ourfindings also suggest the involvement of the actin cytoskeletonin the process and the recruitment of glutamate transporters toactin-rich domains. It has been proposed that the surfacedensity of EAAC1 is controlled by rapid constitutive cyclingbetween the plasma membrane and intracellularcompartments, with the proportions at the cell surface and inendosomal compartments depending on the relative rates oftransporter insertion or removal from the plasma membrane(Gonzalez et al., 2007). Several factors can modify the rate ofinsertion and removal of transporters, among them activationof signaling pathways (e.g., PKC) and interaction with accessoryproteins (Gonzalez and Robinson, 2004a). A possibility is thatALLO, via PKC, may promote the delivery and the fusion ofEAAC1 containing vesicles to the plasma membrane (Gonzalezet al., 2007). In line with this hypothesis, we found thatincubation with monensin (a blocker of exocytosis from therecycling compartment) completely prevented the ALLO-induced EAAC1 surface upregulation. Our data on cytochalasinD treatments also support the possibility that ALLO maypromote the selective binding of EAAC1 to proteins whichtether and anchor the transporter to the actin cytoskeleton,thus limiting its lateral mobility and its recruitment intoendocytic compartments. Interestingly, it has been recentlyreported the interaction of EAAC1 andGLASTwith proteins ofthe NHERF family (Sullivan et al., 2007; D’Amico et al., 2010),whose recruitment to the actin cytoskeleton is controlled byPKC phosphorylation (Brone and Eggermont, 2005). Becauseproteins of the NHERF family have been detected in Schwanncells (Gatto et al., 2003), further research is necessary todetermine whether ALLO may change the glutamatetransporter surface activity by modulating its binding tointeracting partners.

Functional role of glutamate transporters inSchwann cell biology

Pharmacological, electrophysiological andimmunohistochemical evidences indicate that glutamatemay beconsidered as the principal neurotransmitter in many sensoryneurons of the dorsal root ganglia (Yoshimura and Jessell, 1990;Keast and Stephensen, 2000; Carlton, 2001; Huettner et al.,2002). Glutamate has also been proved to be a signalingmediator between the active axon and its surrounding Schwanncells (Lieberman et al., 1989). Therefore, glutamatetransporters on Schwann cells might control the periaxonalglutamate concentration and its cell signaling. Ourimmunofluorescence data on rat sciatic sections showing anenrichment of the EAAC1 staining in the periaxonal domain arein line with this possibility (Fig. 2D).

Glutamate, via activation of glutamate metabotropicreceptors, has been recently involved in the control of twoprocesses critical for the development and regeneration ofperipheral nerves, that is, Schwann cell myelination andproliferation (Garbay et al., 2000; Saitoh and Araki, 2010).

Fig. 8. TIRFM imaging of EGFP–EAAC1 after ALLO treatment.A: ALLO incubation tethers EAAC1 to the plasmamembrane, at thetips of Schwann cell processes. EGFP–EAAC1 transfected Schwanncells were incubated for 30min with 10nM ALLO alone or in thepresence of 2mM cytochalasin D (CytoD). Cells were fixed, and thedistribution of EGFP–EAAC1 in Schwann cells was analyzed byepifluorescence andTIRFmicroscopy. Bar: 10mm.B:ALLO increasesthe plasma membrane expression of EAAC1. Time-lapse imaging ofEGFP–EAAC1intheplasmamembranebymeansofTIRFmicroscopy.The representative image sequence shows a Schwann cell expressingthe EGFP–EAAC1 transporter in the presence of 10 nMALLO (upperpart); the time of the individual frames is indicated. Arrows indicate avesicle, positive for EAAC1,which approaches the plasmamembraneand fuses with it. Arrowheads indicate the recruitment of EAAC1 tothe cell tip. Bar: 5mm. The averaged fluorescence intensity curves ofEGFP–EAAC1 in thepresence (triangles)or absence (circles)of 10 nMALLO showing the percentage of EAAC1 detected at the cell surfaceby TIRFM are reported in the lower part. Data are expressed as apercentageofthe initialaverage intensity (F0)andplottedagainsttime(min). Each point is the mean valueWSEM of fluorescence intensitymeasured on up to 12 different cells in 3 independent experiments(P<0.001). C: ALLO treatment modifies the cell surface profile ofSchwann cells. Cells were treated with or without 10nM ALLO andimaged by TIRFM for 6min. The last photogram is shown. The whiteline indicates the cell profile in the first photogram. Double-arrowsindicate the enlargement in Schwann cell morphology. Bar: 5mm.


1748 P E R E G O E T A L .

Therefore, glutamate transporters by regulating the level ofextracellular glutamate may participate in such a control. Giventhis hypothesis, the EAAC1-mediated glutamate transportupregulation induced by ALLO, would decrease theextracellular glutamate concentration available formetabotropic receptor activation, thus decreasing the Schwanncell proliferation. Surprisingly, we found that ALLO upregulatesSchwann cell proliferation, and this effect is mediated by theEAAC1 activity (Fig. 10). It is therefore possible that ALLOmayregulate cell proliferation by a different pathway. Indeed, theincreased EAAC1 surface expression may also account forenhanced intracellular glutamate accumulation and glutamate isan important intermediate in several metabolic processes.Particularly relevant for Schwann cell physiology may beglutamate involvement in the synthesis of GABA, which hasbeen proposed as an important modulator of Schwann cellproliferation andmyelination (Magnaghi et al., 2009). In linewiththis possibility, it has been recently demonstrated that Schwanncells possess the biosynthetic machinery able to synthesize andrelease GABA in vitro (Magnaghi et al., 2010). Glutamatetransporters may thus provide Schwann cells with glutamatewhich serves as precursor of GABA. It is interesting tounderline that the ALLO effects on glutamate uptake (Fig. 4),

GAD synthesis and GABA release (Magnaghi et al., 2010),indeed, display identical time-dependency, reaching anoverlapping peak after 30–40min of treatment. The hypothesisof a relationship between glutamate uptake and GABAsynthesis in glial cell is further consistent with recent findingsdemonstrating that activation of glutamate transporters resultsin increased GABA release (Heja et al., 2009).

On the basis of our evidences and literature data, thefollowing scenario can be envisaged (Fig. 11): ALLO throughGABA-A receptor causes glutamate uptake upregulation (PKCmediated) andGAD synthesis (via PKA and cAMPmechanisms)(Magnaghi et al., 2010). In the cytoplasm, increased glutamateandGADaccumulationwould in turn promoteGABA synthesisand release. In the extracellular milieu, increased glutamateuptake would reduce the glutamate concentration. Our datasuggest a role of ALLO and glutamate transporters in thecontrol of Schwann cell proliferation but further experimentsare required in order to clarify the significance of glutamate andGABA balance on Schwann cell biology.

Given the involvement of glutamate excitotoxicity in anumber of neurological disorders including peripheralneuropathies (Watkins, 2000; Zhang et al., 2002; Arkaravichienet al., 2003; Bacich et al., 2005), and the recent evidencesindicating a role of glutamate and GABA in the control of

Fig. 9. ALLO increases the EAAC1 delivery to the plasma membrane. A: Monensin treatment prevents the ALLO effect on [3H]D-aspartateuptake upregulation. Schwann cells were incubated with monensin (20mM, 30min) in the absence (CTR) or presence (ALLO) of 10 nM ALLObefore [3H]D-aspartate uptake measurements. Data are expressed as cpm/well/15min represent the meanWSEM of three independentexperiments performed in quadruplicate (MP<0.05 vs. CTR). B: Time-lapse TIRFM imaging of EGFP–EAAC1 in the presence ofmonensin and/orALLO. EGFP–EAAC1 transfected Schwann cells were imaged with 20mM monensin and/or 10nM ALLO for 6min by TIRFM. The first (0minALLO) and the last (6min ALLO) photograms of representative images are shown on the left. Bar: 5mm. The averaged fluorescence intensitycurves of EGFP–EAAC1 in the absence (circles) or presence (triangles) of 10nM ALLO showing the percentage of EAAC1 detected at the cellsurface by TIRFM are reported in the right part. Data are expressed as a percentage of the initial average intensity (F0) and plotted against time(min). Each point is the mean valueWSEM of fluorescence intensity measured on up to 10 different cells in three independent experiments. C:Dynasore does not prevent theALLO-mediated effect. Schwann cellswerepreincubatedwithDynasore (80mM, 1h) and then treatedwith 10 nMALLO in the same solution for 30min, before [3H]D-aspartate uptakemeasurements in the presence of NaCl. D: Time-lapse TIRFM imaging ofEGFP–EAAC1 in thepresenceofDynasore and/orALLO.EGFP–EAAC1transfectedSchwanncellswerepretreatedwithDynasore (80mM,37-C,1h)and imagedwithoutorwith10 nMALLOfor6minbyTIRFM.Thefirst and the lastphotogramsof representative imagesare shownon the left.Arrowheads indicatetherecruitmentofEAAC1tocell tips.Bar:5mm.Theaveragedfluorescence intensitycurvesofEGFP–EAAC1intheabsence(circles) or presence (triangles) of 10 nMALLOshowing the percentage of EAAC1detected at the cell surface byTIRFMare reported in the rightpart (P<0.001).



Schwann cell proliferation and myelination in regenerativeprocesses, the modulation of the Naþ-dependent high affinityglutamate transporters might represent a possible therapeuticapproach for treating peripheral neuropathies and forpromoting regenerative processes.

Acknowledgments

We are grateful to Astrid Wilan for assistance with themanuscript editing. The financial support of the Italian Ministryof education (PUR 2007/2008 to C.P. and V.M.), AssociationFrancaise contre lesMyopathies (AFMGrant no. 14163/2009 toV.M.) and Compagnia Di San Paolo (Bando ProgrammaNeuroscienze 2008 - Project MOVAG) (Grant no. 2229/2008to V.M.) are gratefully acknowledged. We would like to thankProf. Paola Conti and Prof. Carlo de Micheli for providing theHIP-A compound. There is no conflict of interest for the resultspresented in this manuscript.

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Fig. 11. Glutamate and GABA in Schwann cells. We propose anautocrine loop involving glutamate in theGABA signaling of Schwanncells. Indeed, our data on EAAC1 presence support a GABA-Amediated regulation of Schwann cells by GABA and/or theneurosteroid ALLO (Magnaghi et al., 2009). ALLO via GABA-Areceptor activation causes respectively glutamate uptake (PKCmediated) and GAD upregulation (PKA mediated; Magnaghi et al.,2010). In the cytoplasm, glutamate accumulation and GAD activitymight promote GABA synthesis and release. Moreover, increasedglutamate uptake might reduce the extracellular glutamateconcentrations, which together with GABA, may act autocrinally onSchwanncellsdifferentiationandproliferation (Magnaghi et al., 2009).Other mechanisms such as EAAC1 involvement in glutathionemetabolism (Aoyama et al., 2006) should not be excluded.

Fig. 10. Schwann cell proliferation in presence of ALLO andEAAC1-modulators. Schwann cell proliferation after incubation with10 nM ALLO was monitored in the presence or absence of theglutamate transporter-inhibitors Hip-A (10mM) and TBOA (10mM).The BrdU (100mM) was added to the culture medium for the final24 h, then the cells were fixed in PBS-PAF 4%, incubated with theanti-BrdU mouse monoclonal antibody and revealed byimmunofluorescence. MP<0.05 versus control (cells grown in theabsenceofALLO).Data aremeanWSEMof experimentperformed intriplicate.


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Neurosteroid allopregnanolone regulates EAAC1-mediated glutamate uptake and triggers actin changes...

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