Neuropharmacology 79 (2014) 506e514

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Agonist-induced down-regulation of AMPA receptors in oligodendrocyte progenitors Shireen Hossain, Hsueh-Ning Liu, Gabriela Fragoso, Guillermina Almazan* Department of Pharmacology and Therapeutics, McGill University, Montreal, Quebec, Canada

a r t i c l e i n f o

a b s t r a c t

Article history: Received 2 December 2011 Received in revised form 9 December 2013 Accepted 23 December 2013

Prolonged exposure of oligodendrocyte progenitor cultures to non-toxic concentrations of glutamate receptor agonists for 24 h decreased cellular proliferation mediated by a-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid (AMPA) receptors. Since prolonged agonist stimulation can regulate the expression of various families of receptors, we examined this possibility. Pretreatment of progenitor cultures with 100 mM kainic acid (KA) for 1e24 h caused a time-dependent decrease in AMPA receptor activity, determined by agonist-induced 45Ca2þ uptake. The maximum effect (70e80% decrease), observed in the 24 h-pretreated cells, was accompanied by a significant reduction in AMPA receptor subunits, as determined by Western blotting. GluR2/3 and GluR4 subunits were the most affected. Receptor down-regulation and 45Ca2þ uptake were only partially reversible upon KA removal. Furthermore, 24 h co-treatment of cultures with CNQX blocked the KA-induced decreases in calcium uptake. To address whether calpain, a calcium-activated protease, was implicated in the regulation of the AMPA receptor subunits, cultures were treated with the specific inhibitor PD150606 alone or in combination with KA for 24 h. Calpain inhibition significantly increased GluR1 in both conditions and partly reversed downregulation of GluR4 by KA. Collectively, these results indicate that calpain is not involved in the agonist-induced down-regulation of AMPA receptors subunits 2/3 in oligodendrocyte progenitors, while it downregulates GluR1 and GluR4. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: AMPA receptors Extracellular calcium Glutamate Receptor downregulation Kainate Calpain Oligodendrocytes

1. Introduction

a-amino-3-hydroxy-5-methyl-4The expression of isoxazolepropionic acid (AMPA) receptors in oligodendrocytes, the myelin-forming cells of the central nervous system, is well established. Glutamate and related agonists depolarize oligodendrocytes and their progenitors through ionotropic AMPA/KA as well as NMDA receptors (Borges et al., 1994; Gallo et al., 1994b; Holzwarth et al., 1994; Karadottir et al., 2005; Micu et al., 2006; Wyllie et al., 1991). Examination of glutamate receptor currents in single cells revealed different affinity for agonists and desensitization kinetics, suggesting the co-expression of functional AMPAand KA-preferring receptors (Gallo et al., 1994b; Patneau et al., 1994). Calcium imaging experiments, in antigenically identified cultured oligodendrocytes progenitors, further supported the electrophysiological studies showing that activation of AMPA or KA receptors produced large and transient increases in [Ca2þ]i * Corresponding author. Department of Pharmacology and Therapeutics, McGill University, 3655 Promenade Sir William Osler, Room 1321, Montreal, Quebec H3G 1Y6, Canada. Tel.: þ1 514 398 6222; fax: þ1 514 398 6690. E-mail address: [email protected] (G. Almazan). 0028-3908/$ e see front matter Ó 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.12.020

(Holtzclaw et al., 1995; Holzwarth et al., 1994; Meucci et al., 1996; Pende et al., 1994). Furthermore, molecular analysis of glutamate receptors in oligodendroglial lineage cells has demonstrated the expression of AMPA- and KA-preferring subunit mRNAs and proteins in rat, mouse and humans with some species difference (Brand-Schieber and Werner, 2003; Gallo et al., 1994b, 1994a; Garcia-Barcina and Matute, 1996; Holzwarth et al., 1994; Jensen and Chiu, 1993; Kukley and Dietrich, 2009; Newcombe et al., 2008; Patneau et al., 1994; Wosik et al., 2004; Yoshioka et al., 1995). The presumed precursors of the oligodendrocyte lineage, the proteoglycan NG2þ-expressing cells also express the ionotropic AMPA/KA receptors (Kukley and Dietrich, 2009; Lin et al., 2005). They form functional synapse-like associations with neurons in the developing and adult CNS, as shown using immunohistochemical, electrophysiological and ultrastructural studies (Bergles et al., 2000; Lin and Bergles, 2004; Lin et al., 2005). NG2þ cells receive glutamatergic and GABA (g-aminobutyric acid)-ergic synaptic input from neurons (Bergles et al., 2000; Karadottir et al., 2005; Kukley et al., 2008; Lin and Bergles, 2004; Lin et al., 2005), while astrocytes also communicate with NG2þ cells by releasing glutamate (Hamilton et al., 2010). Furthermore, transition of NG2progenitors to the premyelinating stage oligodendrocytes was

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paralleled by rapid removal of synaptic input as well as by downregulation of AMPA and NMDA receptors (De Biase et al., 2010). In fact, induction and expression of longterm potentiation in neuronNG2þ synapses involve the activation of AMPA receptors (Ge et al., 2006). In addition, electrical stimulation of mouse oligodendrocyte progenitor-dorsal root ganglion neurons inducing vesicular glutamate release resulted in the formation of cholesterol-rich signaling domains, Fyn kinase signaling, as well as MBP protein translation (Wake et al., 2011). However, the local synthesis of MBP appeared to be dependent on NMDA but independent of AMPA receptor activation in this system. Prolonged activation of KA-responsive non-NMDA receptors can mediate excitotoxicity in cells of the oligodendroglial lineage (Yoshioka et al., 1995), involving cytosolic calcium overload, mitochondria damage, bax and calpain activation (Sanchez-Gomez et al., 2011). AMPA receptor expression in progenitors was suggested to account for the observed vulnerability to hypoxice ischemic injury or glutamate receptor-mediated excitotoxicity (Itoh et al., 2002; Rosenberg et al., 2003), as postulated for periventricular leukomalacia, the predominant hypoxiceischemic lesion afflicting white matter of premature infants (Back, 2006; Deng et al., 2008; Follett et al., 2004). In addition, sustained activation of AMPA receptors in oligodendrocyte progenitors, mediated by the desensitization blocker cyclothiazide (Berger, 1995; Patneau et al., 1994; Yamada and Tang, 1993), involved massive calcium influx, free radical generation, JNK activation, GSH depletion, and caspase activation (Liu et al., 2002) and cell death. In contrast, chronic exposure of oligodendrocyte progenitors to the AMPA/KA receptor agonist KA in the absence of cyclothiazide and in the presence of PDGF-AA and high concentrations of insulin, two pro-survival factors, was not toxic (Liu and Almazan, 1995; Liu et al., 2002) and inhibited their proliferation through activation of AMPA/KA receptors (Gallo et al., 1996; Liu and Almazan, 1995), while expression of the immediate early genes NGFI-A (Gallo et al., 1994b; Pende et al., 1994) and c-fos (Liu and Almazan, 1995; Pende et al., 1994) were increased. Since many of these studies involved longterm exposure (w24 h) of the cultures to agonist, it was of interest to determine whether AMPA receptor expression was regulated and its functional consequences. The objective of the present study was to determine whether AMPA receptors in oligodendrocyte progenitors are downregulated by prolonged exposure to KA and its functional consequences. We found that pre-exposure of oligodendrocyte progenitors to non-toxic concentrations of agonists but not antagonists, timedependently decreased calcium uptake (AMPA receptor activity). This was accompanied by a most significant reduction in the expression levels of calcium-permeable AMPA receptor subunit proteins. Furthermore, GluR subunits appeared to be differentially regulated by the calcium-dependent cysteine enzyme, calpain. 2. Materials and methods 2.1. Materials Dulbecco’s modified Eagle medium/Ham’s F12 medium (DMEM/F12), Ham’s F12 medium, DMEM, phosphate buffered saline (PBS), 7.5% bovine serum albumin fraction V, penicillin/streptomycin were from Invitrogen (Burlington, ON, Canada). Poly-D-lysine, poly-L-ornithine, Triton X-100, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-tetrazolium bromide (MTT), human transferrin, insulin, and HEPES were from SigmaeAldrich (Oakville, ON, Canada), forskolin, putrescine, and HEPES were from Sigma-Aldrich (Oakville, ON, Canada). Platelet derived growth factor-AA (PDGF-AA) and basic fibroblast growth factor (bFGF) were from PeproTech Inc (Rocky Hill, NJ, USA). The 45Ca2þ and 3H-thymidine isotopes were purchased from Amersham (GE Lifesciences) (Baie d’Urfe, QC, CA). The protein assay kit was from BIO-RAD (Mississauga, ON, Canada). 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX), 6,7-dinitroquinoxaline-2, 3-dione (DNQX), KA, glutamate, 2-amino-3-hydroxy-5methyl-4-isoxazole propionic acid (AMPA) were purchased from Sigma-RBI (Natick, MA, USA). X-OMAT Kodak x-ray film was purchased from Eastman Kodak (Rochester, NY, USA). Primary anti-glutamate subunits (GluR1, GluR2, GluR2/3 and

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GluR4) antibodies were gifts from Dr. Robert Wenthold; goat anti-b-actin antibodies from Santa Cruz Biotechnology (Santa Cruz, CA, USA) or mouse anti-b-actin was from Pierce (Thermo Scientific (Rockford, IL, USA); Horseradish peroxidase (HRP)conjugated secondary antibodies were from Jackson Immunoresearch Laboratories (Cedarlane, Hornby, ON) or BIO-RAD Canada (Mississauga, ON). All other reagents were obtained from VWR (Mont-Royal, QC) or Fisher Scientific (Ottawa, ON, Canada). 2.2. Methods 2.2.1. Cell culture Oligodendrocyte progenitors were isolated from 10-d mixed glial cultures containing astrocytes and microglia that had been prepared from brains of newborn SpragueeDawley rats as described (Almazan et al., 1993). Oligodendrocyte progenitors were subsequently plated on poly-D-lysine-coated culture dishes and expanded in proliferating serum-free medium (SFM) consisting of a DMEM-F12 mixture (1:1), 10 mM HEPES, 0.1% bovine serum albumin, 25 mg/ml human transferrin, 30 nM triiodothyronine, 20 nM hydrocortisone, 20 nM progesterone, 10 nM biotin, 5 mg/ml insulin, 16 mg/ml putrescine, 30 nM selenium. SFM was supplemented with 2.5 ng/ml each of PDGF-AA and bFGF to maintain progenitors proliferation. Cultures were characterized immunocytochemically with cell type-specific antibodies (Cohen and Almazan, 1994; Radhakrishna and Almazan, 1994). More than 95% of the cells were positive for monoclonal antibody A2B5, a marker for oligodendrocyte progenitors in culture while less than 5% were galactocerebrosidepositive oligodendrocytes, glial fibrillary acidic protein-positive astrocytes or complement type-3-positive microglia. For differentiation of oligodendrocyte progenitors, the mitogens PDGF-AA and bFGF were removed from the culture medium and grown in SFM media supplemented with 3% newborn calf serum. All experiments were approved by the McGill Faculty of Medicine Animal Care Committee. 2.2.2. Drug treatments To induce AMPA receptor downregulation, the agonists KA (100 mM), AMPA (100 mM) or glutamate (100 mM) and antagonists CNQX (25 or 50 mM) or DNQX (50 mM) were added to the culture medium (glutamine-free DMEM, supplemented with SFM components, 0.7% BSA, 5 ng/ml PDGF-AA) for 1e24 h. When combining agonists and blockers, antagonists were added 5 min prior to stimulation with AMPA or KA receptor agonists. The calpain inhibitor PD150606 (1e60 mM) was supplemented 30 min before glutamatergic receptor agonists. 2.2.3. 45Calcium uptake functional assay The influx of 45Ca2þ into oligodendrocyte progenitors was determined according to the protocol previously described (Liu et al., 1997). Cells grown overnight in the presence of KA to downregulate the receptors were first washed twice with HBSS and further preincubated for 30 min in Mg2þ-free Locke’s solution (154 mM NaCl, 5.6 mM KCl, 3.6 mM NaHCO3, 1.3 mM CaCl2, 5.6 mM D-glucose, 5 mM HEPES pH 7.4) at 37  C. Stimulation was achieved by replacing incubation solution with 0.5 mL Locke’s solution containing 0.5 mCi 45Ca2þ and the various pharmacological agents tested as indicated in the results section. After 5 min, the cells were washed three times with ice-cold buffer (154 mM choline chloride, 2 mM EGTA, and 10 mM HEPES, pH 7.4) then lysed in 0.1 M NaOH/0.1% Triton-X-100. Radioactivity in the total cell lysate was determined using liquid scintillation counting and corrected for total protein (as determined using BioRad Bradford protein assay). 2.2.4. Western blot analysis Cells were harvested in RIPA (150 mM sodium chloride, 50 mM Tris (pH 8.0), 0.5% sodium deoxycholate, 0.1% SDS, 1% Triton X-100). The protein concentration of samples was determined with the Bio-Rad protein assay. Protein extracts (w30 mg) from the total cellular lysate were resolved using SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membranes by standard protocols. Blots were blocked with 5% dry milk in Tris-buffered saline containing 0.1% Tween20 and then incubated with primary antibodies. Nitrocellulose membranes were incubated with the appropriate HRP-conjugated secondary antibodies and immunoreactive bands visualized by enhanced chemiluminescence and quantified by densitometry using AlphaEaseFC software. To normalize for equal loading and protein transfer, the membranes were reprobed with antibodies for b-actin. Crude membrane preparations of cultured oligodendrocyte progenitors were prepared from 2  10 cm diameter culture dishes (w5e10  106). Cells were washed twice with PBS and scraped off the dish in 2 ml PBS. Following centrifugation at 1500 rpm for 10 min at 4  C, the cell pellet was resuspended in lysis buffer (10 mM TriseHCl, pH 7.4, 5 mM EDTA, 15 mM NaCl, 1% Triton X-100) with mechanical force using a 1 ml pipette to lyse cells and kept on ice for 15 min. The homogenate was centrifuged at 12,000 rpm for 20 min at 4  C. Protein concentration was determined in the supernatants and samples adjusted with loading buffer; Samples were resolved on 7% SDS-PAGE gels. 2.2.5. Mitochondrial activity as an index of cell viability Cell viability was assessed using the MTT assay that measures mitochondrial dehydrogenase activity, according to the protocol described previously (Mosmann, 1983). The assay detects cleavage of 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-

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tetrazolium bromide MTT by active mitochondria in viable cells to an insoluble formazan product. Oligodendrocyte progenitor cultures were incubated with 0.125 mg/ml MTT at 37  C for 3 h. The medium was then collected, centrifuged to pellet any floating viable cells and the formazan crystals in both the plate and pellet were solubilized in an acideisopropanolol mixture. Absorbance was measured at 595 nm with a microplate spectrophotometer (Bio-Rad). 2.2.6. Statistical analysis Results are expressed as mean  SEM (Standard Error of mean). Statistical comparisons were made by either unpaired Student’s t-test followed by Bonferroni correction, or by ANOVA Dunnett’s or TukeyeKramer post hoc test to identify differences between treatments. p values 90% decrease), followed by GluR2 (w80%), GluR4 (w80%) and GluR2/3 (w80%) subunits levels. We concurrently assessed the accumulation of two proteins found in myelin expressed by differentiated oligodendrocytes e myelin basic protein (MBP) and 20 ,30 -cyclic-nucleotide 30 phosphodiesterase (CNP) to determine the degree of maturation in culture (Fig. 1b). Hence, there is a developmental regulation of the expression of AMPA receptor subunits in oligodendrocytes, as reported by another group (Itoh et al., 2002). For all subsequent experiments, which required longer treatment with pharmacological agents, oligodendrocyte progenitors were maintained in the presence of PDGF-AA (5 ng/ml) and high insulin to prevent their differentiation and promote their survival.

agonist-induced manner (Fig. 2b).

45

Ca2þ-uptake in a concentration-dependent

3.3. Diminished AMPA receptor activity following chronic agonist exposure We previously demonstrated that longterm (1e24 h) exposure of oligodendrocyte progenitors to AMPA receptor agonist KA (100 mM) in the presence of growth factors and in the absence of cyclothiazide was not cytotoxic (Liu and Almazan, 1995; Liu et al., 2002) (Supplementary Fig. S2 and Table 3). Following agonist stimulation, AMPA receptors characteristically undergo rapid desensitization (Michaelis, 1998) and, may undergo downregulation with continued stimulation. In order to study the effect of longterm stimulation of AMPA receptors, oligodendrocyte progenitors were pre-treated with KA for 1e24 h and receptor activity assessed by 45Ca2þ uptake. Cells were washed twice before a 5-min stimulation with KA (100 mM) in the presence of cyclothiazide (25 mM). As shown in Fig. 3a, chronic agonist stimulation reduced 45 Ca2þ uptake in a time-dependent manner. Calcium uptake decreased slightly after 1 h (w10% from maximum), reaching a statistically significant reduction (w40%) after 8 h, with a maximal (90%) after 24 h of pretreatment with KA. Similar to KA,

3.2. Expression of functional AMPA receptors in oligodendrocyte progenitors Next, we determined the functionality of AMPA receptors in oligodendrocyte progenitors. As AMPA receptor activity can be assessed by measuring calcium uptake (Liu et al., 1997), cells were stimulated for 5 min with increasing concentrations of the glutamatergic receptor agonists AMPA and KA (1e1000 mM) in the presence of cyclothiazide (25 mM), an allosteric modulator that blocks the rapid desensitization of AMPA receptors to potentiate the response. Both agonists dose-dependently increased 45Ca2þuptake (Fig. 2a). Pre-treating oligodendrocyte progenitors with CNQX (1e 50 mM), a competitive AMPA/KA receptor antagonist, blocked

Fig. 1. Developmental regulation of AMPA receptor subunits in oligodendrocytes. Oligodendrocyte progenitor cultures were allowed to differentiate for 0, 3 and 6 days and then harvested for Western blot analysis. a, the relative levels of AMPA receptor subunits GluR1, GluR2, GluR2/3 and GluR4 were determined in samples containing 30, 15, 20, and 20 mg protein, respectively, of the total cell lysate from oligodendrocytes. Membranes were reprobed for b-actin as a measure of equal loading control. Relative protein levels of each subunit were quantified and expressed as a percentage of control (day 0) after normalizing for loading (actin). b, Myelin basic protein (MBP) and 20 ,30 cyclic-nucleotide 30 -phosphodiesterase (CNP) levels were assessed in the samples as a marker of differentiation. Shown are representative blots of a representative experiment performed in triplicate. ap < 0.05, bp < 0.01, cp < 0.001, One way ANOVA, Dunnett’s post-hoc test.

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pretreatment of oligodendrocyte progenitors with glutamate (100 mM) or AMPA (100 mM) for 24 h also decreased the subsequent response to KA and cyclothiazide stimulation (w30e70%), while 24 h pre-treatment with the antagonists CNQX (50 mM) or DNQX (50 mM) alone did not affect the posterior response to a 5 min KA þ cyclothiazide stimulated 45Ca2þ uptake. However, the decreased calcium uptake caused by KA was fully prevented by the presence of CNQX during the pretreatment period (Table 1). 3.4. Receptor subunit protein downregulation following sustained AMPA receptor activation Since there was a decline in calcium uptake following chronic agonist exposure, we next determined whether continuous (18 or 24 h) exposure of oligodendrocyte progenitor cultures to KA (100 mM) in the absence of cyclothiazide altered protein levels of AMPA receptor subunits (GluR1-4). Indeed KA pre-treatment decreased all AMPA receptor subunit levels, as assessed in total cell lysate (Fig. 3b). To study the effect of KA pre-treatment on cell membrane levels of individual AMPA receptor subunits, we conducted cellular subfractionation and compared the crude membrane (M) and total (T) cell fractions by immunoblotting (Fig. 3c). KA pretreatment comparably reduced AMPA subunit levels in the membrane fraction. Thus, for all future experiments assessing subunit expression, we used the total cell lysate to minimize manipulation error. Interestingly, KA pre-treatment appeared to exhibit subunit-selective downregulation, where GluR2 was the least affected (decreased by w20%), while GluR2/3 and GluR4

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subunits were the most affected (decreased by w40 and 70%, respectively) (Fig. 3c). The observed decreases were of similar magnitude both in the crude membrane preparations and whole cell lysates. These results suggest that different kinetics of downregulation exist for each AMPA subunit. In contrast, chronic (24 h) treatment of oligodendrocyte progenitors with the AMPA/KA antagonist CNQX did not affect the relative protein levels of any of the GluR subunits (Table 2). Thus, indicating a selective downregulation of AMPA receptor subunits GluRs 1, 3 and 4 resulting from continuous agonist-stimulation. 3.5. Partial recovery of AMPA receptor activity and subunits expression following chronic activation We next determined whether the decreased receptor activity induced by continuous KA exposure was reversible following agonist removal. Oligodendrocytes progenitors pre-exposed to 100 mM KA for 24 h were washed and allowed to recover for 2e24 h, at which time calcium uptake was evaluated by a 5 min exposure to KA (100 mM) and cyclothiazide (25 mM). Indeed, there appeared to be a partial time-dependent functional recovery of the AMPA receptors from 4 h (w30%), with a maximal recovery occurring at 24 h (w73%) (Fig. 4a). We also examined whether AMPA receptor subunit protein levels would recover following KA washout using Western blotting analysis. Oligodendrocyte progenitors were pre-treated for 24 h with KA, washed and allowed to recover for 2, 4, and 24 h. Total cellular extract showed that the small decrease in GluR2 levels appeared to recover by 4 h post-KA exposure (Fig. 4b). 3.6. A role for calpain in AMPA receptor subunit downregulation

Fig. 2. Dose-dependent increase in calcium uptake in oligodendrocyte progenitors following AMPA or KA exposure. a, 45Ca2þ-uptake was assessed in oligodendrocyte progenitors stimulated with either AMPA (filled square) or KA (open triangle) (1e 1000 mM) in the presence of cyclothiazide (25 mM) and 45Ca2þ for 5 min. Cells were washed, harvested, and 45Ca2þ-uptake was subsequently determined. Results shown are the mean  SEM dpm per well in 5 min (corrected for basal stimulation) of three independent experiments performed in quadruplicate. As compared to basal uptake levels: p < 0.05 (50 mM KA), p < 0.01 (100 mM KA), p < 0.001 (50, 100, 500 and 1000 mM AMPA; 500 and 1000 mM KA), One way ANOVA, Dunnett’s post-hoc test. In b, cells were pretreated for 5 min with 1e50 mM CNQX, then stimulated with either KA (100 mM) or AMPA (100 mM) in the presence of cyclothiazide (25 mM) and 45Ca2þ for 5 min. Cells were subsequently washed, harvested, and calcium uptake determined. Results shown are the mean  SEM of the maximum stimulation (%) for three independent experiments performed in quadruplicate. As compared to maximum uptake levels: p < 0.05 (5 mM CNQX þ KA), p < 0.01 (10, 25 and 50 mM CNQX þ KA), p < 0.001 (1, 2.5, 5, 7.5, 10, 15 and 30 mM CNQX þ AMPA), One way ANOVA, Dunnett’s post-hoc test.

The C-terminal domain of GluR1 was shown to be a substrate of calpain (Bi et al., 1996), a calcium-dependent cysteine protease responsible for the degradation of many cellular proteins. Members of this family have been implicated in a number of neurodegenerative processes (Chan and Mattson, 1999) and in AMPA receptormediated excitotoxicity in oligodendrocyte progenitors (Liu et al., 2002). Thus, the possible role of calpain in the longterm agonistinduced downregulation of AMPA receptors in oligodendrocyte progenitors was next examined. Progenitor cultures were treated with the non-competitive calpain-selective pharmacological inhibitor PD150606 (30 mM) prior to 18 h KA treatment (100 mM) and the four AMPA receptor subunits were evaluated using immunoblotting (Fig. 5). Note that treatment with KA (100 mM) or PD150606 (30 mM) was not toxic to progenitors, as MTT reduction (survival) was not affected (Table 3). Interestingly, PD150606 treatment alone increased GluR1 by 30% and prevented the downregulation induced with chronic KA treatment. Calpain inhibitor appears to reduce the downregulation of GluR4 induced by KA. In contrast, GluR2 and GluR2/3 protein levels were not affected by the treatment. From these experiments, we conclude that calpain does not appear to be involved in regulation of GluR2 and GluR2/3 subunits, while GluR1 and to lesser extent GluR4, are downregulated by calpain in oligodendrocyte progenitors as reported in other cellular systems (Bi et al., 1996; Bi et al., 1998b; Bi et al., 1998a; Bi et al., 2000; Lu et al., 2001; Yuen et al., 2007b, 2007a). 4. Discussion In this study, the effect of chronic AMPA receptor agonist- and antagonist-stimulation on receptor activity and subunit expression in oligodendrocyte progenitors was explored. Using calcium uptake assays and Western blot analyses, it was found that pretreatment of

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Fig. 3. Chronic exposure of oligodendrocyte progenitors to KA leads to a timedependent decrease in AMPA receptor-mediated calcium uptake and the downregulation of AMPA receptor subunits. a, oligodendrocyte progenitors were first pretreated with 100 mM KA for 1, 6, 8, 12, 18 and 24 h in serum-free media containing PDGF. Cells were then washed twice to remove KA, and stimulated for 5 min with KA (100 mM) and cyclothiazide (25 mM) in the presence of 45Ca2þ. Cells were washed, harvested and the calcium uptake determined in cell lysates. Results are expressed as a percentage of maximum stimulation; the mean  SEM of three independent experiments performed in triplicate are shown. All values were normalized according the amount of protein per well. In b and c, oligodendrocyte progenitors were pretreated with 100 mM KA (Pre-KA) for 18 h in serum-free media containing PDGF then harvested for Western blotting. The protein levels of AMPA receptor subunits (GluR1 (R1), GluR2 (R2), GluR2/3 (R2/3) and GluR4 (R4)) were assessed in both the total cell lysate (T) (b, c) and membrane fraction (M) (c) in control and pre-KA-treated cultures. Actin was used to determine equal loading. Shown are representative Western blots for total cell lysates in b, and the mean  SEM of at least three independent experiments performed in triplicate are illustrated in c. ap < 0.05, bp < 0.01, cp < 0.001, One way ANOVA, Dunnett’s post-hoc test.

oligodendrocyte progenitors with the receptor agonists KA and AMPA, but not the AMPA/KA receptor antagonists CNQX and DNQX, resulted in diminished receptor activity and a downregulation of AMPA receptor subunits, which affected mostly GluR3 and R4. Finally, the results point to the involvement of the calciumactivated protease calpain in AMPA receptor downregulation in a subunit-specific manner. In our study, differentiation of oligodendrocyte progenitor, starting from day 3, decreased the expression of all four AMPA

receptor subunits (Fig. 1), as others reported for 4-d differentiated cultured oligodendrocytes (Itoh et al., 2002). Similarly, metabotropic glutamate receptors were expressed at high levels in oligodendrocyte progenitors, and were downregulated with differentiation (Deng et al., 2004). Collectively, the developmental downregulation of AMPA and metabotropic glutamate receptors may explain why oligodendrocyte becomes resistant to glutamate receptor-mediated insults such as hypoxiaeischemia following maturation (Kavanaugh et al., 2000; McDonald et al., 1998). In fact, AMPA receptors in oligodendrocyte progenitors were found to be much more permeable to calcium than pre-progenitors, immature and mature oligodendrocytes (Itoh et al., 2002). Under our culture conditions, presence of PDGF-AA and high levels of insulin, treatment with glutamate or AMPA receptor agonists at 100 mM does not produce toxicity but decreases proliferation of oligodendrocyte progenitors (Gallo et al., 1996; Liu and Almazan, 1995). This observation was also made in cerebellar granule cell cultures (Resink et al., 1995, 1996). Thus, the allosteric modulator cyclothiazide, which blocks the rapid desensitization of AMPA receptors, is required to induce cell death in progenitors. Therefore, in the current study, we have maintained progenitors in the presence of PDGF-AA and high concentrations of insulin, which can activate IGF-I receptors, and protect oligodendrocyte progenitors from glutamate-mediated excitotoxicity (Ness et al., 2004). We have found that sustained agonist stimulation of AMPA receptors with KA downregulated AMPA receptor subunits at the protein level, and largely prevented calcium uptake, an indicator of receptor activity. Similar results were obtained with AMPA and glutamate pretreatment, which significantly decreased the calcium-uptake induced by 5 min treatment with KA and cyclothiazide. In contrast, prolonged exposure of oligodendrocyte progenitors to the AMPA/KA receptor antagonists CNQX and DNQX altered neither AMPA receptor subunit protein expression levels nor receptor activity. The most pronounced effect of agonist-induced downregulation was on GluR subunits 3 and 4, which are highly calcium-permeable and more abundantly expressed than GluR1. While only a small reduction in GluR1 and GluR2 levels was observed. The molecular composition of AMPA channels, assembled from GluR1e4, determines their permeability to calcium. Thus, AMPA receptors containing GluR2 have limited calcium permeability compared to those lacking GluR2 (Geiger et al., 1995; Pellegrini-Giampietro et al., 1997; Sattler and Tymianski, 2001). The downregulation of AMPA receptor proteins in oligodendrocyte progenitors was accompanied by a large decrease in calcium uptake, reflecting the greater abundance of GluR2 as compared to the calcium-permeable subunits. The changes in subunit levels in the total cellular extracts followed a similar trend as that observed in the crude cell membrane fractions. In recovery experiments, KA was removed from the culture medium and progenitor cells were maintained for 2e24 additional hours to determine whether the changes in receptor activity and protein levels were reversible. The longer time allowed calcium uptake to be restored by w70%, suggesting a significant recovery of calcium-permeable subunits. Furthermore, Western blot analysis of individual subunits indicated a tendency towards recovery between 20 and 50% for the calcium permeable subunits GluR3 and GluR4. Our results suggest that the relatively high decrease in calcium permeable AMPA receptor subunits in oligodendrocyte progenitors following continuous exposure to agonist could be a potentially protective mechanism to combat a posterior cytotoxic insult as has been proposed for their rapid desensitization under normal physiological conditions (Itoh et al., 2000; Kavanaugh et al., 2000; Liu et al., 2002; Matute et al., 1997; Sanchez-Gomez and Matute, 1999; Sanchez-Gomez et al., 2003; Yoshioka et al., 1995). In oligodendrocyte progenitors, excitotoxicity was shown to be mediated

S. Hossain et al. / Neuropharmacology 79 (2014) 506e514 Table 1 Regulation of 45Ca2þ uptake stimulated by KA þ cyclothiazide in oligodendrocyte progenitors pre-treated with agonist and antagonists. Treatment

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Ca2þ uptake (nmol/min/mg protein)

Basal Stimulated (KA þ CYZ) Pretreat-AMPA Pretreat-KA Pretreat-Glu Pretreat-CNQX Pretreat-DNQX Pretreat-CNQX þ KA

4.9 60.6 24.1 15.3 45.8 55.9 54.7 70.3

       

0.4 1.6*** 1.2a 1.3a 2.1b 5.2 1.6 1.0 (n ¼ 1)

% of KA þ CYZ stimulation (nmol/min/mg protein) e 100 34 18 76 91 89 116

Progenitors were pretreated with KA (100 mM), AMPA (100 mM), glutamate (Glu) (100 mM), CNQX (50 mM), or DNQX (50 mM), or CNQX and KA (50 mM þ 100 mM) for 24 h and washed 4 times prior to 5 min stimulation with KA and cyclothiazide (100 þ 25 mM). Values are mean  SEM of one or three independent experiments performed in triplicate. Statistical differences : ***p < 0.001 compared with basal; a p < 0.001 compared with KA and cyclothiazide; bp < 0.05 compared with KA and cyclothiazide, cp < 0.001, Oneway ANOVA, t-test with Bonferroni correction.

by a subset of AMPA receptors that are GluR2-lacking and calciumpermeable (Deng et al., 2006). From immunoprecipitation studies, at least 20% of the total GluR3 and 4 appear to co-assemble in progenitors, and this subpopulation could be responsible of cytotoxicity (Itoh et al., 2002). Interestingly, preconditioning oligodendrocyte progenitors with sub-lethal oxygen-glucose deprivation led to the downregulation of GluR2, increased AMPA receptor calcium permeability and increased cell death (Deng et al., 2003). Using the pharmacological inhibitor PD150606, our results suggest that the calcium-activated protease calpain is involved in the downregulation of AMPA subunits GluR1 and GluR4, but not GluR2 and GluR3. The effects on GluR1 corroborate previous reports showing that prolonged treatment of neuronal cultures with glutamate agonists suppressed AMPA-mediated currents, and decreased surface and total receptor expression, which were blocked by calpain inhibitors (Bi et al., 1996; Bi et al., 1998b; Bi et al., 1998a; Bi et al., 2000; Lu et al., 2001; Yuen et al., 2007b, 2007a). AMPA GluR1 subunit was shown to be a substrate of calpain, truncating the C-terminal tail of the protein (Lu et al., 2000). This regulation was dependent on the phosphorylation state of the GluR1 subunit by calcium-calmodulin-dependent protein kinase II (Yuen et al., 2007a). Downregulation of GluR1 was proposed to provide a negative feedback by calpain to reduce excitotoxicity under pathological conditions such as ischemia and epilepsy (Yuen et al., 2007a). In our study, we found that treatment of oligodendrocyte progenitors with the calpain inhibitor PD150606 alone resulted in the upregulation of GluR1, as compared to control. This result is of interest since expression of GluR1 but not GluR2 was found to be upregulated in oligodendrocytes in active multiple sclerosis lesion borders (Newcombe et al., 2008). These authors suggested that upregulation of GluR1 may render oligodendrocytes vulnerable to excitotoxic damage by glutamate, thereby preventing Table 2 Regulation of AMPA receptors expression in oligodendrocyte progenitors pretreated with agonist and antagonist.

GluR1 GluR2 GluR2/3 GluR4

Control (% of CO)

KA (% of CO)

CNQX (% of CO)

132.0 1015.0 305.0 330.5

96.3 825.0 173.3 104.8

141.5 1077.0 254.1 274.9

(100.0) (100.0) (100.0) (100.0)

(73.0) (81.3) (56.8) (31.7)

(107.2) (106.1) (83.3) (83.2)

Progenitors were treated with KA (100 mM) or CNQX (50 mM) for 24 h before harvesting. Total cell lysates were analyzed using Western blotting. Shown are the mean optical densities from two independent experiments performed in duplicate. The means expressed as a percentage of control (CO) are in parentheses.

511

remyelination in multiple sclerosis plaques. With respect to GluR4, we are unaware of another study reporting on its regulation by calpain. Our results with PD150606, which reverses KA-induced downregulation, strongly suggest that similar to GluR1, it is regulated by this protease and would merit further analysis. The developmental downregulation of AMPA receptor subunits in oligodendrocyte progenitors and that induced by long-term KA exposure, may involve the regulation of several subunit-interacting proteins. AMPA receptor intracellular trafficking and localization are regulated through its interactions with distinct scaffolding PDZ domain proteins, including glutamate receptor-interacting protein 1 (GRIP1) (Liu and Cull-Candy, 2005; Steiner et al., 2005), PSD-95 (Cai et al., 2006), synapse-associated protein 97 (Howard et al., 2010) and Pick1 (Hanley, 2008), as has been extensively studied in synaptic membranes. Expression or degradation of these

Fig. 4. Functional recovery of oligodendrocyte progenitors following chronic exposure to KA. Oligodendrocyte progenitors pre-treated (Pre-KA) or not with 100 mM KA for 24 h in serum-free media containing PDGF were washed and allowed to recover for 2e 24 h. In a, AMPA receptor functional recovery was assessed using 45Ca2þ uptake following stimulation with 100 mM KA and 25 mM cyclothiazide. The mean  SEM of three independent experiments performed in triplicate are illustrated. Statistical differences compared with non-KA pre-treated stimulated cultures: ap < 0.001; compared with Pre-KA: *p < 0.05, **p < 0.01 and ***p < 0.001, Oneway ANOVA, TukeyeKramer post-hoc test. In b, the total cell extracts were used to determine the relative expression levels of AMPA receptor subunits (GluR1 (R1), GluR2 (R2), GluR2/3 (R2/3) and GluR4 (R4)) by immunoblotting. The mean optical densities from a representative experiment of two performed in duplicate are illustrated in b. In c, the protein concentration (mg/mL) in the samples under the different experimental conditions was determined. Shown in the graph are the mean  SEM from three independent experiments performed in triplicate. Statistical differences were not significant, Dunnett’s post-hoc test.

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S. Hossain et al. / Neuropharmacology 79 (2014) 506e514 Table 3 Inhibition of calpain with PD150606 does not affect oligodendrocyte progenitor survival. Treatment

% of control

Control 100 mM Kainate

100.00  18.72 121.08  6.28

Control 1 mM PD150606 3 mM PD150606 30 mM PD150606 60 mM PD150606

100.00 96.20 120.88 102.80 101.16

    

4.15 19.31 4.20 6.67 3.48

Cell survival was assessed using an MTT reduction assay in which oligodendrocyte progenitors were treated with kainate (100 mM) or PD150606 (1e60 mM) for 24 h. All data shown are mean  SEM of one to five independent experiments performed in quadruplicate.

Pick1, which in neurons was reported to affect GluR2/3 expression (Iwakura et al., 2001). Studies on NG2þ oligodendrocyte progenitors showed that GRIP1 acts as a scaffolding molecule clustering NG2 and GluR2 (Stegmuller et al., 2003) and suggested that this protein complex may participate in glialeneuronal recognition and signaling. Therefore, it would be of interest to determine whether GRIP1 and/or other PDZ containing proteins are regulated in the same manner as AMPA receptor subunits. In summary, we report that calcium-permeable AMPA receptors are selectively downregulated by longterm agonist stimulation and this involves calpains for the GluR1 and GluR4 subunits. The profound downregulation in GluR3 and GluR4 renders the receptors impermeable to calcium. This mechanism may be used by oligodendrocyte progenitors as a protective mechanism against excitotoxicity. Acknowledgments Fig. 5. Differential regulation of AMPA receptor subunit expression by calpain. The regulation of AMPA receptor subunits by calpain was evaluated using the pharmacological inhibitor PD150606 (PD). Oligodendrocyte progenitor cultures were pre-treated with 30 mM of PD150606 for 1 h before addition of 100 mM KA. Cells were maintained for 18 h then lysed. AMPA receptor subunit levels were assessed in whole cell lysates. Representative Western blots from one of three experiments performed in triplicate are shown in a. Actin was used to correct for equal loading. The quantifications for GluR1-4 from three independent experiments are shown in the graphs below (bee), expressed as a percentage of control. Statistical significance was determined using Oneway ANOVA with Bonferroni correction: as compared to control: ap < 0.05, b p < 0.01, cp < 0.001; or as compared to KA: *p < 0.05, **p < 0.01.

We would like to dedicate this article to the memory of Dr. Robert J. Wenthold for his seminal work on the biochemistry of glutamate receptors and his generosity for providing AMPA selective antibodies, which made this work possible. We would like to thank Jun Fang for his help with the preparation of some of the oligodendrocyte cultures. The authors acknowledge the Multiple Sclerosis Society of Canada (MSSC) and Canadian Institute of Health Research (CIHR) for operating grants to G.A. and MSSC studentships to Hsueh-Ning Liu and Shireen Hossain. Appendix A. Supplementary data

proteins may be affected with chronic AMPA receptor activation by agonists. For instance, prolonged stimulation of hippocampal slices with the ampakine CX617 resulted not only in a downregulation of GluR1, GluR2/3, but also in the degradation of their anchoring proteins SAP97 and GRIP1 (Jourdi et al., 2005). The downregulation and slow recovery of these AMPA receptor subunits were attributed to similar changes in SAP97 and GRIP1. Thus, these may be interesting proteins to assay for the future. For instance, GRIP, an AMPA receptor anchoring protein of GluR2, was shown to be a substrate of calpain (Lu et al., 2001). Several other members of the PDZ family of proteins, PSD-95 and the cytoskeletal protein spectrin are also truncated by calpain (Baudry and Lynch, 1981; Lu et al., 2000, 2001). Thus, in addition to playing a role in the downregulation of GluR2, calpain may be indirectly involved in regulating the expression of GluR2 by acting on several modulators, thereby affecting receptor protein interaction and translocation and hence, ultimately AMPA receptor activity. Another associated protein which may be affected by PD150606 treatment is the PDZ molecule,

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Agonist-induced down-regulation of AMPA receptors in oligodendrocyte progenitors.

Prolonged exposure of oligodendrocyte progenitor cultures to non-toxic concentrations of glutamate receptor agonists for 24 h decreased cellular proli...
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