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NEUROPHARMACOLOGY

Prolonged Positive Modulation of -Amino-3-hydroxy-5-methyl-4-isoxazolepropionic Acid (AMPA) Receptors Induces Calpain-Mediated PSD-95/Dlg/ZO-1 Protein Degradation and AMPA Receptor Down-Regulation in Cultured Hippocampal Slices

Neuroscience Program and Department of Biology (H.J., X.L., M.B.), University of Southern California, Los Angeles, California; and Departments of Psychiatry and Human Behavior (H.J., T.Y., X.B.) and Anatomy and Neurobiology (J.C.L., C.M.G.), University of California Irvine, Irvine, California

Received January 17, 2005; accepted March 18, 2005.

	Abstract

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Prolonged exposure of cultured hippocampal slices to CX614 [2H,3H,6aH-pyrrolidino[2'',1''-3',2']1,3-oxazino[6',5'-5,4]-benzo[e]1,4-dioxan 10-one], a positive -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor (AMPAr) modulator, decreases receptor response to synaptic stimulation, an effect that could reflect reduced receptor expression. The present study investigates this down-regulation and its underlying mechanisms using cultured rat hippocampal slices. Chronic treatment with CX614 gradually reduced levels of glutamate receptor (GluR)1 and GluR2/3 AMPAr subunits and of their anchoring proteins synapse-associated protein 97 (SAP97) and glutamate receptor interacting protein 1 (GRIP1) through 48 h. Decline in SAP97 and GRIP1 levels was associated with increased abundance of lower molecular weight bands, suggesting degradation of these proteins. CX614 effects were partially reversible after drug removal. GluR1 and GluR2/3 down-regulation and their slow recovery were associated with similar changes in SAP97 and GRIP1 levels. Treatment with CX614 for 48 h significantly reduced AMPAr mRNA levels in hippocampus, whereas 8-h exposure did not. Blockade of ionotropic glutamate receptors prevented CX614-induced decrease in AMPAr subunits and mRNA, with regional selectivity, although an AMPAr blocker was more efficacious than an N-methyl-D-aspartate receptor blocker. Blockade of calpain activity reduced CX614-induced degradation of SAP97 and GRIP1 and prevented decreases in AMPAr subunit but not mRNA levels. Treatment with CX614 alone or in combination with glutamate receptor blockers or calpain inhibitor III did not modify lactate dehydrogenase release into culture medium, implying the absence of cell toxicity. We conclude that CX614-induced AMPAr protein loss is primarily mediated by AMPAr activation and involves calpain-dependent proteolysis of SAP97 and GRIP1. CX614-induced suppression of AMPAr gene expression is, however, calpain-independent, and all these effects are not associated with cell damage.

CX614 belongs to the benzamide family of positive AMPA receptor modulators (or ampakines). These drugs up-regulate AMPAr-mediated electrophysiological responses to glutamate, increase miniature excitatory postsynaptic current frequency and amplitude, and facilitate LTP (Arai et al., 2000, 2002a,b; Suppiramaniam et al., 2001). CX614 increases the amplitude of AMPAr-mediated currents, prolongs the "open-channel" state of the receptor, and increases the frequency and amplitude of miniature excitatory postsynaptic potential. However, prolonged exposure to CX614 results in desensitization and/or rundown of the receptor response (Arai et al., 2000). Such AMPAr down-regulation could possibly reflect enhanced AMPAr internalization and/or removal of the receptors from the synaptic pool.

Calpain is a Ca²⁺-dependent protease present in the synaptic compartment. Its activation is required for the induction of LTP and occurs after incubation of neurons with NMDA, glutamate, and theta burst stimulation (Oliver et al., 1989; Bednarski et al., 1995; Muller et al., 1995; Vanderklish et al., 1996). Conversely, suppression of calpain expression or activity blocks LTP (Oliver et al., 1989; Denny et al., 1990; Bednarski et al., 1995; Vanderklish et al., 1996). Moreover, calpain truncates the C-terminal domains of several AMPA and NMDA receptor subunits, as well as PSD-95 and GRIP1, members of the PDZ family of proteins (Lu et al., 2000, 2001), and the cytoskeletal protein spectrin (Baudry and Lynch, 1981). In a recent report, we showed that prolonged incubation of cultured hippocampal slices with CX614 resulted in calpain activation and calpain-mediated proteolysis of spectrin (Jourdi et al., 2005). Together, these studies indicate that calpain plays a critical role in the reorganization of glutamatergic postsynaptic compartment after a number of stimuli, resulting in increased calcium concentrations in postsynaptic structures. They also suggest that calpain-mediated proteolysis of AMPAr-associated PDZ proteins might be involved in down-regulation of AMPAr. The possibility that prolonged exposure of cultured hippocampal slices to CX614 could lead to the destabilization of AMPAr complexes and the elimination of the receptor subunits was addressed in the present study.

	Materials and Methods

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Hippocampal Slice Cultures. Ten- to 12-day-old rat pups were obtained from The Jackson Laboratory (Bar Harbor, ME) and used to prepare hippocampal slice cultures as described previously (Jourdi et al., 2005). Briefly, animals were decapitated, and the brains were dissected, trimmed, and 400-µm-thick hippocampal slices were cut using a McIllwain tissue slicer (Fotodyne Inc., New Berlin, WI). Slices were explanted onto Millipore insert membranes, placed in six-well culture plates, and fed with regular slice culture medium (50% basal Eagle's medium, 25% Earl's balanced salt solution, 136 mM sodium chloride, 2 mM calcium chloride, 2.5 mM magnesium sulfate, 5 mM sodium bicarbonate, 3 mM glutamine, 40 mM glucose, 0.5 mM ascorbic acid, 20 mM HEPES buffer, 1 mg/l insulin, 5 U/ml penicillin, and 5 mg/l streptomycin; pH 7.3) supplemented with 5% fetal bovine serum and 5% horse serum. Slices were kept in culture for 2 weeks and fed with 1 ml of fresh medium every other day. Slices were then treated with CX614, glutamate receptor antagonists, or calpain inhibitor III, at the following concentrations: CX614, 50 µM; AP-5, 100 µM; CNQX, 50 µM; and calpain inhibitor III, 10 µM. At the indicated times after treatment initiation, slices were collected and processed for immunoblotting, immunocytochemistry, or in situ hybridization as described below. The AMPAr-positive modulator CX614 was generously provided by Cortex Pharmaceuticals (Irvine, CA). AP-5, CNQX, and calpain inhibitor III were purchased from Sigma-Aldrich (St. Louis, MO).

Immunocytochemistry. Slices were washed twice in cold phosphate-buffered saline, fixed overnight with 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, at 4°C, postfixed and cryoprotected overnight at 4°C in 4% paraformaldehyde in 0.1 M phosphate buffer (PB) supplemented with 20% sucrose, and then sectioned using a freezing microtome. Free-floating sections (25 µm) were washed in 0.1 M PB and blocked for 1 h with 10% horse or goat serum in 0.1 M PB (+ 1% Triton X-100) and then incubated with the primary antibody (1:1000) overnight at 4°C in 5% horse serum. Tissue was then washed in 0.1 M PB and incubated with the biotinylated secondary antibody (diluted 1:400) in 5% horse or goat serum/0.1 M PB for 2 h at room temperature. After washes in 0.1 M PB, the tissue was incubated in avidin biotin complex working solution (ABC Elite Vectastain kit; Vector Laboratories, Burlingame, CA) and then reacted using the peroxidase substrate SK-4100 Vector kit (Vector Laboratories) with 3,3'-diaminobenzidine as chromagen. After final washes in 0.1 M PB, the sections were mounted onto Superfrost Plus slides (Fisher Scientific, Tustin, CA), air-dried, dehydrated with ethanol, cleared with Americlear (Fisher Scientific, Tustin, CA), and coverslipped. Stained sections were examined using a Zeiss Axioskop2 microscope fitted with an AxioCam camera and operated with AxioVision 3.1.2.1 [EC] software (Carl Zeiss, Thornwood, NY).

Polyacrylamide Gel Electrophoresis and Western Blotting. Slices were collected into cold homogenizing buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 10 mM NaF, and 1 mM Na₃VO₄; all from Sigma-Aldrich, St. Louis, MO) supplemented with protease inhibitor cocktail (Sigma-Aldrich) [2.08 mM 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 1.6 µM aprotinin, 40 µM leupeptin, 80 µM bestatin, 30 µM pepstatin A, and 28 µM trans-3-carboxyoxirane-2-carbonyl-L-leucylagmatine], sonicated five times using a Virsonic cell disruptor set at 50% for 4- to 7-s intervals, and protein concentrations were determined. Equal amounts (20–40 µg) of sample proteins were separated according to their molecular weight on 8 to 10% SDS polyacrylamide gels and transferred onto nylon/polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA). After transfer, membranes were blocked with 5% milk in 1x TBS-T (137 mM NaCl, 20 mM Tris base, and 0.1% Tween 20; pH 7.4) for 1.5 h. Membranes were probed with the primary antibody diluted with 5% milk (1: 1000) and incubated overnight at 4°C. Membranes were then washed with 1x TBS-T and incubated with horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences Inc., Piscataway, NJ) in a 1:10,000 dilution with 1x TBS-T for 1 h followed by washing in TBS-T. Membranes were exposed to enhanced chemiluminescence Western blotting detection reagent (Amersham Biosciences Inc.) for 2 min, placed in an autoradiography cassette, and exposed to Hyperfilm (Amersham Biosciences Inc.) for different lengths of time. All membranes were stripped and reprobed with anti-actin antibodies (Sigma-Aldrich) as loading controls. Intensities of immunoreactive bands were quantified by densitometry, normalized to actin content, and analyzed by Student's t test.

Antibodies. For Western blotting, the following primary antibodies were used at the indicated final concentrations: 0.1 µg/ml monoclonal mouse anti-actin (Sigma-Aldrich), 0.5 µg/ml polyclonal rabbit anti-GluR1 (Chemicon International, Temecula, CA), 0.5 µg/ml polyclonal rabbit anti-GluR2/3 (Chemicon International), 0.5 µg/ml polyclonal rabbit anti-NR1 (Chemicon International), 0.5 µg/ml polyclonal rabbit anti-NR2A/B (Chemicon International), polyclonal rabbit anti-GRIP1 (Upstate Biotechnology, Chicago, IL), monoclonal mouse anti-SAP97 (StressGen Biotechnologies, Victoria, BC, Canada). Horseradish peroxidase-conjugated secondary antibodies were purchased from Amersham Biosciences Inc. and used at 1:10,000 dilution. For immunocytochemistry, the polyclonal anti-GluR1 and anti-GluR2/3 antibodies and a monoclonal mouse anti-GluR2 (Chemicon International) antibody were used. All antibodies were diluted to 5 to 10 µg/ml. Secondary antibodies for immunocytochemistry were purchased from Vector Laboratories.

In Situ Hybridization. After drug treatments, cultures were washed twice with Tris-buffered saline and fixed overnight in cold fixative solution (4% paraformaldehyde solution in 0.1 M PB; pH 7.3). Cultures were kept in fixative at 4°C until sectioning at 25 µm with a freezing microtome. Sections were mounted onto Superfrost Plus slides and processed for the in situ hybridization localization of GluR1 and GluR2 mRNAs using ³⁵S-labeled complementary RNA probes at a concentration of 1 x 10⁷ cpm/ml with hybridization incubation times of 16 to 20 h at 60°C as described previously (Lauterborn et al., 2000). The GluR1 and GluR2 complementary RNAs were transcribed from BglI and BamHI digests of p59/2 and pRB14, respectively, and are complementary to 895 and 900 bases, respectively, of the 3' ends, including the noncoding region. After hybridization, the tissue was treated with RNase A (20 µg/ml; 30 min; 45°C) and washed in descending concentrations (2x–0.1x) of saline sodium citrate buffer, 30 min each (1x = 0.15 M NaCl/0.015 M sodium citrate; pH 7.0). The tissue was air-dried and processed for Biomax film (Eastman Kodak, Rochester, NY) autoradiography with exposure times of 1 to 2 days. Hybridization densities within hippocampal CA1 and CA3b stratum pyramidale were measured from film autoradiograms, with labeling densities calibrated relative to film images of ¹⁴C-labeled standards (American Radiolabeled Chemicals, St. Louis, MO) using the AIS imaging system (Imaging Research, St. Catherines, ON, Canada). For each field, significance of effect of treatment was determined by the two-way analysis of variance followed by planned post hoc analysis using Student-Newman-Keuls post hoc test for individual comparisons. Statistical analyses were conducted using Prism software (version 3; GraphPad Software, Inc., San Diego, CA), and the 95% confidence level was considered significant.

Lactate Dehydrogenase Activity. Lactate dehydrogenase (LDH) enzymatic activity in the culture medium was used to evaluate the extent of cellular damage produced in cultured slices subjected to the different treatments. Briefly, culture medium was collected after 8- or 48-h incubation of slices with CX614 alone, calpain inhibitor III alone, the glutamate receptor blockers AP-5 and CNQX, CX614 + glutamate receptor blockers, CX614 + calpain inhibitor III, and from untreated control cultures. Spectrophotometric determination was used to measure LDH activity by monitoring the rate at which the substrate pyruvate is reduced to lactate (Regan and Choi, 1994). Reaction mixtures consisted of 600 µl of potassium phosphate buffer (0.1 M; pH 7.5 at 25°C), 400 µl of supernatant culture medium, and 500 µl of sodium pyruvate solution (22.7 mM in 0.1 M phosphate buffer; pH 7.5). The absorbance was read at 340 nm at 30-s intervals for 3 min. All reagents were purchased from Sigma-Aldrich.

	Results

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Treatment of Cultured Hippocampal Slices with CX614 Induces a Gradual Loss of AMPAr Subunits. Hippocampal slices obtained from postnatal day 10 rat pups were maintained in culture for 10 to 14 days. Previous studies by our laboratories indicated that treatment of cultures for 48 h with CX614 decreased AMPAr subunit mRNA and protein levels (Lauterborn et al., 2003). To assess the time course of CX614 on AMPAr protein, cultured slices were incubated with 50 µM CX614 for 0, 4, 8, 12, 24, and 48 h (Fig. 1). Significant decreases in GluR1 and GluR2/3 levels were observed after 4 h of incubation. There was a near linear decline in GluR1 and GluR2/3 subunits over time with CX614 treatment through 24 h (Fig. 1B). The decrease in AMPAr subunit levels continued at a slower rate between 24 and 48 h of treatment. Probing the same samples with antibodies specific to the NMDA receptor subunits NR1 and NR2A/B did not show significant differences from control at any time point (Fig. 1C); levels of NR1 subunits were decreased by 15%, whereas those of NR2A/B were increased by 5% after 48-h treatment with CX614 (Fig. 1D). The results for GluR1 and GluR2/3 subunits were confirmed using immunohistochemistry; untreated control cultures and cultures treated with CX614 for 48 h were stained with the following AMPAr-specific antibodies: anti-C-terminal GluR1, anti-C-terminal GluR2/3, and anti-N-terminal GluR2 (Fig. 1E). Overall, immunostaining with all three antibodies was decreased. In addition, with antibodies against the C-terminal domain of GluR1, intense staining of some cell bodies was observed, an effect reminiscent of what we previously observed after calcium treatment of tissue sections (Bi et al., 1998).

View larger version (41K): [in this window] [in a new window]   Fig. 1. CX614-induced down-regulation of AMPAr in cultured hippocampal slices. Cultures were incubated in the absence (DMSO vehicle control) or presence of 50 µM CX614 and processed for immunoblotting or for immunostaining. A, representative blots of samples from cultures incubated continuously for 48 h with the vehicle control or with 50 µM CX614 for 4, 8, 24, or 48 h labeled with anti-GluR1 or anti-GluR2/3 antibodies. B, quantification of the levels of GluR1 and GluR2/3 from four duplicate experiments (total n = 8 samples per time point). *, p < 0.05; **, p < 0.01; ***, p < 0.001, versus control levels, Student's t test. C, representative blots of samples from control cultures or cultures treated with 50 µM CX614 for 48 h labeled with anti-NR1 or anti-NR2A/B antibodies. D, quantification of results derived from four duplicate experiments did not show significant differences in NR1 and NR2A/B protein levels compared with controls. Dashed line indicates protein expression in vehicle control samples. E, photomicrographs showing immunostaining with AMPAr-specific antibodies in region CA1 of cultured hippocampal slices either untreated (left column) or treated with CX614 for 48 h (right column). Sections were incubated with anti-C-terminal GluR1, anti-C-terminal GluR2/3, or anti-N-terminal GluR2 antibodies. Scale bar, 100 µM.

View larger version (21K): [in this window] [in a new window]   Fig. 2. Effects of CX614 on AMPAr-associated PDZ proteins. Cultured hippocampal slices were incubated with 50 µM CX614 or vehicle (control) for 48 h and processed for immunoblotting with SAP97 and GRIP1 antibodies. A, representative blots for SAP97 and GRIP1. B, quantification of major band densities obtained in four duplicate experiments (total n = 8 samples/group) for SAP97 and GRIP1. Dashed line indicates protein expression in vehicle control samples. C, representative immunoblots showing increased intensities of lower molecular weight bands (arrows) reacting with SAP97 and GRIP1 antibodies in CX614-treated samples. D, quantification of lower molecular weight species from eight experiments for SAP97 and GRIP1 degradation products. Dashed line indicates protein expression in vehicle control samples. *, p < 0.01; **, p < 0.005; Student's t test.

Parallel Effects of CX614 on AMPAr Subunits and SAP97 and GRIP1. Incubation of cultured hippocampal slices with CX614 induced a marked decline in AMPAr subunit levels within 8 h. To determine whether these effects of CX614 were reversible, cultures were treated with CX614 for 8 h and then transferred to control medium and maintained for various lengths of time (0, 4, 8, 12, 24, and 48 h). In parallel, sister cultures were exposed continuously to CX614 for 48 h. Cultures were collected and levels of AMPAr subunits, SAP97, and GRIP1 were analyzed by Western blotting. As expected, treatment of cultures with CX614 for 8 h induced a significant decrease in levels of AMPAr subunits and SAP97 (Fig. 3). Similarly, slices treated continuously with CX614 for 48 h exhibited a large decrease in levels of AMPAr subunits and of their partner PDZ proteins. Transfer of cultures to fresh CX614-free medium was not sufficient to immediately reverse the effects of the drug, as levels of AMPAr subunits, SAP97, and GRIP1 continued to decline for another 4 to 8 h. Levels of AMPAr subunits and of their partner PDZ proteins had slightly recovered by 24 h of drug washout. However, even after 48 h of incubation in the absence of CX614, levels of AMPAr subunits, SAP97, and GRIP1 did not completely return to control levels.

View larger version (19K): [in this window] [in a new window]   Fig. 3. Time course for the recovery of AMPAr and PDZ protein expression. Bar graphs showing protein levels for GluR1 and SAP97 (A) and GluR2/3 and GRIP1 (B) in cultured hippocampal slices that were either treated with 50 µM CX614 for 8 h and then transferred to CX614-free fresh culture medium (wash) and collected various times thereafter (open symbols) or treated continuously with CX614 for 48 h (solid symbols). Data are expressed as percentage of vehicle-control levels and are means ± S.E.M. of three duplicate experiments. *, p < 0.05; **, p < 0.005; , p < 0.05; , p < 0.01, Student's t test.

View larger version (52K): [in this window] [in a new window]   Fig. 4. Effects of CNQX and AP-5 on CX614-induced changes in levels of AMPAr subunits and their associated PDZ proteins. Hippocampal slice cultures were incubated with or without 50 µM CX614 for 48 h and in the presence or absence of 100 µM AP-5 and 50 µM CNQX. A and C, representative Western blots labeled with anti-GluR1, -GluR2/3, -SAP97, or -GRIP1 antibodies under various conditions examining the effects of either coapplication of AP-5 and CNQX (A) or separate application of the blockers (C) on CX614 effects. B, quantification of four duplicate experiments as shown in A with results expressed as percentage of control values. D, quantification of four duplicate experiments as shown in C with results expressed as percentage of control values. *, p < 0.05; **, p < 0.01; , p < 0.05; , p < 0.01; Student's t test; n = 8/group.

Prolonged Exposure of Cultured Hippocampal Slices to CX614 Decreases the Expression of AMPAr Subunit mRNAs. To further understand the mechanisms of CX614-mediated down-regulation of AMPA receptor function, we used in situ hybridization to analyze the effects of prolonged treatment of cultured hippocampal slices with CX614 on GluR1 and GluR2 mRNA levels (Fig. 5). In a first set of experiments, cultured hippocampal slices were incubated under control conditions or with CX614, AP-5 + CNQX, or CX614 together with AP-5 + CNQX for 8 h. Although GluR1 mRNA levels showed a tendency to decrease in CX614-treated slices, no significant differences in GluR1 or GluR2 mRNA levels were observed under any conditions in CA1 and CA3 pyramidal cell layers at this time point (data not shown). In contrast, slices incubated for 48 h with CX614 exhibited a marked decrease in GluR1 and GluR2 mRNA levels in the pyramidal cell layer of both hippocampal subfields (Fig. 5). Treatment with CNQX + AP-5 alone for 48 h had opposite effects on GluR mRNA levels with modest increases in GluR1 (consistent with protein changes presented in Fig. 4) and small decreases in GluR2 mRNA levels: significant effects of treatment were reached for GluR1 and GluR2 mRNA levels in CA3 and CA1, respectively. Interestingly, treatment with CNQX + AP-5 markedly reversed the effects of CX614 for both mRNAs only in CA3. Two-way ANOVA analysis revealed a significant reduction of CX614 effects by the blockers for GluR2 mRNA (p = 0.0006) and a strong trend for GluR1 mRNA (p = 0.0509). By contrast, in region CA1, CNQX + AP-5 treatment did not block the CX614-induced decreases in GluR mRNA levels. Two-way ANOVA analysis revealed no significant reduction of CX614 effects by the blockers for both GluR1 (p = 0.008) and GluR2 (p = 0.0075) mRNAs in this field.

View larger version (45K): [in this window] [in a new window]   Fig. 5. Effects of CNQX and AP-5 on CX614-induced down-regulation of GluR1 and GluR2 mRNA levels. A, photomicrographs of film autoradiograms showing in situ hybridization for GluR1 mRNA in cultured hippocampal slices under control conditions (vehicle) or treated for 48 h with CX614 alone, CNQX + AP-5, or CX614 + CNQX + AP-5 (top). Bar graphs showing quantification of GluR1 cRNA hybridization levels in the pyramidal cell layer of regions CA3 and CA1 obtained in three duplicate experiments (n = 9–14 cultures/group). B, photomicrographs of film autoradiograms showing in situ hybridization for GluR2 mRNA in cultured hippocampal slices under control conditions or treated for 48 h with CX614 alone, CNQX + AP-5, or CX614 + CNQX + AP-5 (top). Bar graphs showing quantification of GluR2 cRNA hybridization levels in the pyramidal cell layer of regions CA3 and CA1 obtained in three duplicate experiments (n = 14–15 cultures/group). *, p < 0.01; **, p < 0.05; ***, p < 0.005; ns, not significant; Student-Newman-Keuls; scale bar, 500 µm.

Effects of a Calpain Inhibitor on CX614-Induced Down-Regulation of AMPAr and Their Interacting PDZ Proteins. As mentioned above, treatment of cultures with CX614 increased the amount of small molecular weight bands that reacted with SAP97 and GRIP1 antibodies. We previously identified GRIP1 and PSD-95, another PDZ protein that shares a similar structure with SAP97 and that interacts with glutamate receptors, as substrates of the calcium-activated protease calpain (Lu et al., 2000, 2001). Preliminary results showed that probing brain membrane fractions pretreated with Ca²⁺ (2 mM and 30 min at 37°C, conditions that activate endogenous calpain) with anti-GRIP1 antibodies showed bands that migrated at the same level as those generated after treatment of cultured hippocampal slices with CX614 (data not shown). Moreover, we recently reported that CX614 treatment activates calpain and leads to the degradation of the cytoskeletal protein spectrin (Jourdi et al., 2005). These results suggested that prolonged treatment with CX614 could result in calpain-mediated SAP97 and GRIP1 degradation, thereby leading to AMPAr down-regulation by interfering with PDZ protein-dependent stabilization and/or intracellular AMPAr trafficking. To test this hypothesis, we incubated cultured hippocampal slices with CX614 with or without calpain inhibitor III for 48 h. Western blot analysis indicated that calpain inhibitor III significantly reduced CX614-induced decreases in AMPAr subunits as well as decreases in the levels of their interacting PDZ proteins (Fig. 6). Again, the results underscored the parallel effects of calpain inhibition on GluR1 and SAP97 and on GluR2/3 and GRIP1. In particular, calpain inhibitor III almost completely reversed the effect of CX614 on GRIP1 and GluR2/3.

View larger version (43K): [in this window] [in a new window]   Fig. 6. Effects of calpain inhibitor III on CX614-mediated down-regulation of AMPAr subunits and their associated PDZ proteins. A, representative immunoblots of samples probed for the total protein expression of GluR1, GluR2/3, SAP97, and GRIP1 in cultured hippocampal slices treated with 50 µM CX614 with or without calpain inhibitor III (Cal Inh III; 10 µM) for 48 h. Samples were obtained from slices treated in the absence of CX614 and calpain inhibitor III (control), CX614 alone, calpain inhibitor III alone (Cal Inh III), or the combination CX614 + Cal Inh III. B, quantification of the immunoreactive bands obtained in four duplicate experiments (total n = 8/group); results are expressed as percentage of the values in control conditions and are means ± S.E.M. Dashed line indicates protein expression levels in control samples. *, p < 0.01; **, p < 0.05; Student's t test.

View larger version (45K): [in this window] [in a new window]   Fig. 7. Effects of calpain inhibitor III on CX614-induced decrease in AMPAr subunit mRNA levels. Control and CX614-treated (50 µM) cultured hippocampal slices were incubated with and without calpain inhibitor III (Cal Inh III; 10 µM) for 48 h. A, photomicrographs showing the in situ hybridization localization of GluR1 mRNA in cultured hippocampal slices under control conditions, or treated for 48 h with CX614 alone, calpain inhibitor III (Cal Inh III) alone, or CX614 + Cal Inh III (top). Bar graphs show quantification of film autoradiograms for GluR1 cRNA densities obtained in three duplicate experiments (n = 12–18 cultures/group). B, photomicrographs showing the in situ hybridization localization of GluR2 mRNA in cultured hippocampal slices under control conditions, or treated 48 h with CX614 alone, calpain inhibitor III (Cal Inh III) alone, or CX614 + Cal Inh III (top). Bar graphs shows quantification film autoradiograms for GluR2 cRNA densities obtained in three duplicate experiments (n = 15–18 cultures/group). *, p < 0.005; ns, not significant; Student's t test; scale bar, 500 µm.

CX614 Effects Are Not Due to Excitotoxicity. LDH activity has been widely used to assess the integrity of the cell membrane and as a measure of toxicity-induced cellular damage that could lead to cell death (Regan and Choi, 1994). To investigate whether the marked decreases in levels and expression of AMPAr subunits and PDZ proteins after prolonged incubation of cultured hippocampal slices with CX614 could result from neuronal toxicity, we measured LDH activity in the culture medium from hippocampal slices treated with the AMPAr modulator under a variety of experimental conditions for 8 and 48 h (Fig. 8). There were no significant differences in LDH activity in cultures treated with CX614 alone, calpain inhibitor III alone, or CNQX + AP-5 at either time point. Similarly, treatment of slices with combinations of CX614 and calpain inhibitor III, or CX614 and AP-5 + CNQX did not modify LDH activity.

View larger version (17K): [in this window] [in a new window]   Fig. 8. Effects of various prolonged treatment with CX614 and other agents on LDH release in cultured hippocampal slices. A and B, slices treated with CX614, CX614 + AP-5 + CNQX, calpain inhibitor III, CX614 + calpain inhibitor III, or left untreated. Incubation medium was collected after 8- or 48-h incubation in the above-mentioned conditions and assayed for LDH activity. Results are expressed as percentage of control values and are means ± S.E.M. of four triplicate (n = 12) experiments.

	Discussion

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Recently, we showed a gradual accumulation of CX614-induced calpain-mediated spectrin breakdown products that was completely blocked by calpain inhibitor III and by the AMPAr blocker CNQX but unaffected by the NMDAr blocker AP-5 (Jourdi et al., 2005). In the present study, decreases in levels of AMPAr subunits and of their respective anchoring PDZ proteins were significant after 4 h of CX614 treatment and became greater with longer incubation intervals. Coadministration of AP-5 and CNQX completely blocked CX614 effects on AMPAr subunits and PDZ proteins. The effects of CX614 on AMPAr and PDZ proteins were partially reduced in the presence of CNQX alone, and AP-5 was less efficacious in blocking CX614 effects on AMPAr and PDZ proteins. Previous studies have demonstrated that NMDArs are tonically active in hippocampal slices (Sah et al., 1989), and our results indicated that AP-5 partially reduced CX614 effects on SAP97 and GluR2/3. In addition, AP-5 significantly decreased CX614 effects on GRIP1 but not on GluR1. Together, our results argue against NMDArs being the principal source of increases in intracellular Ca²⁺ needed for calpain activation with the manipulations described here. However, AMPArs are Na⁺ channels and are, by and large, impermeable to Ca²⁺ ions (Lomeli et al., 1994; Jensen et al., 1998; Carlson et al., 2000; Iizuka et al., 2000; Krampfl et al., 2002; Kumar et al., 2002). Interestingly, elevated intracellular Na⁺ levels can increase Ca²⁺ release from intracellular stores (Hoyt et al., 1998; Zhang and Lipton 1999), thus CX614-induced increases in AMPAr function and the resulting depolarization may contribute to increased intracellular levels of Ca²⁺. Pertinent to this, ryanodine receptors, which regulate intracellular Ca²⁺ release, are also targets for calpain proteolysis (Shoshan-Barmatz et al., 1994). Thus, CX614-dependent increases in AMPAr activity might enhance intracellular Ca²⁺ concentrations and consequently activate calpain that then can truncate ryanodine receptors; this would exacerbate Ca²⁺ release from intracellular stores and further enhance calpain-dependent degradation of AMPAr-interacting PDZ proteins and spectrin. Interestingly, calpain activation, ryanodine receptors, and the release of Ca²⁺ from intracellular stores are tied to aspects of AMPAr function. Calpain activation is required for LTP induction (Vanderklish et al., 1996), and recent studies of type 3 ryanodine receptors provide strong support for the involvement of intracellular Ca²⁺ stores in calpain activation because deletion of this receptor impairs some forms of AMPAr-mediated synaptic plasticity, LTP, and spatial learning (Balschun et al., 1999; Shimuta et al., 2001).

Our results clearly show that calpain activation is predominantly downstream from AMPAr and that it mediates CX614-induced AMPAr protein loss and degradation of PDZ proteins and spectrin (Jourdi et al., 2005). In particular, calpain inhibition blocked CX614-induced decrease in PDZ protein levels and formation of PDZ protein degradation products, and it blocked CX614 effects on AMPAr subunits. In addition, down-regulation of individual AMPAr subunits and their recovery after CX614 washout were associated with similar changes in their interacting PDZ proteins. GRIP1 is implicated in GluR2 stabilization (Jourdi et al., 2003), and GRIP1 truncation by calpain has been shown to decrease GRIP1-GluR2 association (Lu et al., 2001). Our data also indicate that SAP97, which is implicated in GluR1 intracellular trafficking, in its insertion in membranes, and in the stabilization of GluR1 protein (Jourdi et al., 2003; Collingridge et al., 2004), is a calpain substrate as well.

Calpain activation leads to the truncation of GluR1 and GluR2 C-terminal domains (Bi et al., 1998). Thus, a reduction in AMPAr subunits and/or a loss of their C-terminal epitopes could contribute to CX614-induced decreases in AMPAr immunoreactivities observed in this study. Although our immunostaining results cannot exclude truncation of AMPAr C-terminal domains from being responsible for lower levels of immunoreactivity, the results obtained with the anti-N-terminal GluR2 antibody strongly favor the loss of total protein expression with chronic exposure to CX614, at least for GluR2. In addition, truncation of GluR1 and GluR2 C-terminal domains (Bi et al., 1998), and degradation of PDZ proteins and spectrin by calpain might dissociate AMPAr from the cytoskeleton, change their channel properties, and alter their rate of internalization.

AMPAr down-regulation generally involves dissociation from interactions with cytoskeletal and PDZ proteins and is followed by internalization and proteolysis (Ahmadian et al., 2004; Collingridge et al., 2004; Lee et al., 2004). As such, prolonged application of CX614 is likely to cause destabilization and internalization of surface-bound receptors and reduced recycling to the synapse given the down-regulation of SAP97 and GRIP1 and their calpain-mediated degradation observed here. In fact, the interaction of AMPAr with the cytoskeleton and their internalization are highly regulated mechanisms involving several kinases and phosphatases (Iwakura et al., 2001; Rong et al., 2001; Ahmadian et al., 2004; Collingridge et al., 2004). Future studies will investigate whether CX614 and other positive AMPAr modulators recruit distinct kinase pathways to regulate AMPAr surface expression. In addition, various manipulations can induce AMPAr internalization, including LTD or treatment with insulin. The type and duration of the inducing stimulus determine the fate of internalized AMPAr that can be either recycled to the synapse or targeted for terminal proteolysis (Iwakura et al., 2001; Ahmadian et al., 2004; Collingridge et al., 2004; Lee at al., 2004). Future studies will investigate whether LTD-induced AMPAr internalization implicates calpain and whether CX614, and other positive AMPAr modulators, regulate AMPAr surface expression.

BDNF is important for AMPAr expression (Narisawa-Saito et al., 2002; Jourdi et al., 2003), and chronic exposure of hippocampal slice cultures to CX614 initially leads to increases in BDNF mRNA and protein expression that are not maintained past 24 to 48 h. However, intermittent application of CX614 was successfully applied and resulted in sustained increases in BDNF mRNA and protein levels (Lauterborn et al., 2003). Thus, future studies will assess the effects of similar protocols on AMPAr expression, PDZ protein degradation, and calpain activation.

Consistent with the protein data, prolonged exposure to CX614 also affected AMPAr gene expression and reduced their mRNA levels. The loss of GluR mRNA expression occurred within all principal cell layers of hippocampus with 48-h CX614 treatment. Although the combination of AP-5 and CNQX significantly inhibited CX614-induced decreases in total hippocampal AMPAr protein levels, these blockers did not equally inhibit the decrease in GluR mRNA levels between regions CA3 and CA1. AP-5 and CNQX cotreatment blocked the reduction in GluR mRNA levels in CA3, but it was ineffective in CA1. One explanation for this regional disparity may be due to developmental differences in AMPA receptor antagonist binding between the CA3 and CA1 subfields. During postnatal development, [³H]CNQX binding attains adult levels in CA3 by postnatal day 15, whereas in CA1 it is not attained until postnatal day 40 (Standley et al., 1995). Given that the present study utilized slice cultures that at the time of drug treatment approximated postnatal day 20 animals, the deficiency in CA1 [³H]CNQX binding may account for the regional difference in the blocking effect observed here. Alternatively, the differential effects of the combination CNQX and AP-5 on CX614-induced decreases in GluR mRNA levels between the hippocampal subfield might reflect greater spontaneous release of glutamate in CA1 than in CA3.

Overall, the main findings suggest that several factors can contribute to CX614-induced down-regulation of AMPAr. First, enhancing AMPAr activity is a major mechanism underlying AMPAr down-regulation; it involves activity-dependent, calpain-mediated degradation of spectrin and AMPAr-interacting PDZ proteins, which would be expected to interfere with receptor stabilization and trafficking. A second mechanism of AMPAr down-regulation involves calpain-independent decrease of the transcription of the genes coding for these subunits, at least in CA1. Future studies should assess whether other manipulations, which increase synaptic activity in cultured slices (e.g., treatment with functionally distinct ampakines, inhibition of GABAergic transmission), have similar or differential effects on levels of PDZ proteins and AMPAr to those observed here.

In conclusion, this study further implicates calpain in physiological activity-induced changes in postsynaptic structures in the absence of cell toxicity and demonstrates that with prolonged enhancement of glutamatergic synaptic activity, AMPAr loss 1) is mediated by increased AMPAr activation, 2) involves calpain-dependent proteolysis of SAP97 and GRIP1, 3) is reflected at the mRNA level by suppression of AMPAr gene expression, and 4) is not associated with cell damage.

	Acknowledgements

 
We thank Jihua Liu, Anna Marie Tran, Eduardo A. Ramirez, Ulises Martinez, and Yue Quin Yao for technical assistance.

	Footnotes

 
This work was supported by National Institute of Neurological Disorders and Stroke Grant P01NS045260-01A1 (to C.M.G.). Portions of this work have been presented as meeting abstracts (Jourdi et al., Program 895.6. 2003 Abstract Viewer/Itinerary Planner. Society for Neuroscience, Washington, DC, 2003 Online; Jourdi et al., Program 50.16. 2004 Abstract Viewer/Itinerary Planner. Society for Neuroscience, Washington, DC, 2004 Online).

Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.105.083873.

ABBREVIATIONS: AMPA, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid; AMPAr, -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor(s); LTP, long-term potentiation; LTD, long-term depression; PDZ, PSD-95/Dlg/ZO-1; SAP97, synapse-associated protein 97; PSD-95, postsynaptic density protein 95; GRIP1, glutamate receptor interacting protein 1; NMDA, N-methyl-D-aspartate; BDNF, brain-derived neurotrophic factor; GluR, glutamate receptor; CX614, [2H,3H,6aH-pyrrolidino[2'',1''-3',2']1,3-oxazino[6',5'-5,4]benzo[e]1,4-dioxan 10-one]; AP-5, 2-amino-5-phosphonopentanoic acid, CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; PB, phosphate buffer; TBS-T, Tris-buffered saline-Tween 20; LDH, lactate dehydrogenase; DMSO, dimethyl sulfoxide; ANOVA, analysis of variance; NMDAr, N-methyl-D-aspartate receptor.

Address correspondence to: Dr. Michel Baudry, Neuroscience Program, University of Southern California, 3641 W Way, HNB118, Los Angeles, CA 90089-2520. E-mail: baudry{at}usc.edu

	References

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