Neurochemistry International 39 (2001) 151– 160 www.elsevier.com/locate/neuint
Increased ambient glutamate concentration alters the expression of NMDA receptor subunits in cerebellar granule neurons Gvido Cebers *, Aleta Cebere, Attila D. Kova´cs, Helene Ho¨gberg, Tiago Moreira, Sture Liljequist Department of Clinical Neuroscience, Di6ision of Drug Dependence Research, Karolinska Institutet, Karolinska Hospital, Building L1:01, SE-17176 Stockholm, Sweden Received 17 October 2000; received in revised form 22 December 2000; accepted 28 December 2000
Abstract Effects of prolonged (48 h) inhibition of glutamate reuptake on the relative abundance of mRNAs coding for N-methyl-D-aspartate (NMDA) receptor subunits, and the expression of corresponding proteins were investigated in primary cultures of rat cerebellar granule neurons. In cells exposed to the glutamate transport blocker, L-trans-pyrrolidine-2,4-dicarboxylate (PDC), the expression of the C1 exon-positive NR1 mRNA variant was reduced by about 40% whereas, the expression of C1-negative mRNA was increased leading to significant reduction of the + C1/−C1 ratio. The expression of the N1-negative NR1 variants was slightly reduced following exposure to PDC, indicating that increased medium levels of glutamate changed the relative abundance of NR1 splice-variant expression but did not reduce the overall NR1 transcription. Expression of NR2A and NR2B mRNAs was 40–50% lower in PDC-treated cells as compared to control. Immunoblot experiments revealed that PDC exposure reduced the expression of NR1 and all NR2 proteins with NR2A and NR2B proteins being reduced to a greater extent than NR1. The overall decrease in NMDA receptor subunit protein expression was considerably more pronounced than the reduction of their corresponding mRNAs, suggesting involvement of a post-transcriptional regulation. Our data support the hypothesis that functional activity and number of NMDA receptors are regulated by strength of the glutamatergic input. Thus, reduced glutamate uptake resulting in increased concentration of ambient glutamate initiate a series of adaptive responses manifested as a gradual down-regulation of the functional activity and expression of NMDA receptors. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Glutamate transporters; L-trans-pyrrolidine-2,4-dicarboxylate; Immunocytochemistry; RT-PCR; Neurotoxicity; Rat
1. Introduction Glutamate is the major excitatory neurotransmitter in the CNS, where it activates ionotropic receptors that gate ion channels, and metabotropic receptors that act via G-proteins (Monaghan et al., 1985; Collingridge and Lester, 1989; Tanabe et al., 1992; Schoepp and Conn, 1993). Based on their pharmacological properties, ionotropic glutamate receptors have been divided into three sub-classes: N-methyl-D-aspartate (NMDA), a-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA), and kainate receptors (Monaghan et al., * Corresponding author. Tel: +46-8-51775741; fax: + 46-851775231. E-mail address:
[email protected] (G. Cebers).
1985; Boulter et al., 1990; Hollmann and Heinemann, 1994). Stimulation of NMDA receptors increases the cytoplasmic free Ca2 + concentration in neurons (Burgoyne et al., 1988) thereby activating a number of effectors, as e.g. Ca2 + /calmodulin-dependent ion pumps, phosphatases, and protein kinases (Gjertsen and Doskeland, 1995). Since NMDA receptors have a widespread distribution in the brain, high Ca2 + permeability and a unique voltage-dependent activity regulation, they are believed to play a crucial role in the differentiation of neurons during CNS development, and in formation of memory and learning in the adult brain (Mayer and Westbrook, 1987; Burgoyne and Cambray-Deakin, 1988; Nicoll et al., 1988; Collingridge and Singer, 1990; Bliss and Collingridge, 1993). However, excessive acti-
0197-0186/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S0197-0186(01)00014-6
152
G. Cebers et al. / Neurochemistry International 39 (2001) 151–160
vation of NMDA glutamate receptors results in a sustained elevation of free cytoplasmic Ca2 + , that plays a pivotal role in neuronal degeneration and death associated with various CNS disorders (McBean and Roberts, 1985; Collingridge and Singer, 1990; Meldrum and Garthwaite, 1990; Choi, 1992; Orrenius and Nicotera, 1993; Turski and Turski, 1993). Recently we described (Cebers et al., 1999) how prolonged inhibition of high-affinity glutamate reuptake produced by glutamate transport blocker, L-transpyrrolidine-2,4-dicarboxylate (PDC), alters glutamate receptor functions in primary cultures of rat cerebellar granule neurons. We found that PDC-induced glutamate reuptake blockade caused a linear and relatively slow elevation of the medium concentration of endogenously released glutamate, which following 48-h exposure reached about 100 mM, causing immediate neuronal death if applied acutely to intact neurons. Surprisingly, no excitotoxicity could be detected in PDC-exposed neurons, which were almost completely insensitive to exogenously applied NMDA receptor agonists. The magnitude of this functional down-regulation of NMDA receptors was shown to be dependent on the duration of glutamate transport inhibition and was accompanied by a dramatic decrease in the binding capacity of the NMDA receptor antagonist, [3H]MK801, (50% reduction of both the number of binding sites and affinity for [3H]MK-801) demonstrative of pronounced functional and/or structural rearrangement of NMDA receptors. The aim of this study was to examine effects of PDC-induced blockade of glutamate reuptake on the NMDA receptor subunit mRNA concentrations and on the production of the corresponding proteins in cultured rat cerebellar granule neurons. Thus, we have measured the relative abundance of mRNAs encoding for NMDA receptor subunits NR1 (and its splice variants) and NR2A-B by using RT-PCR with primer pairs specific for the aforementioned gene products. We also used immunoblotting and immunocytochemistry for quantification and cellular localisation of NMDA receptor subunit proteins.
2. Experimental procedures
2.1. Cerebellar granule cell cultures Primary cultures of cerebellar granule cells were prepared from 8-day-old Sprague– Dawley rat (B&K Universal, Sollentuna, Sweden) cerebellum as previously described (Cebers and Liljequist, 1995). Briefly, after incubation in 0.25% trypsin solution cells were dispersed by trituration in a DNase and soybean trypsin inhibitor-containing solution (0.01% and 0.05%, respectively) and plated (1×106 cells/ml) onto either glass
coverslips placed in 24-mm culture plates, in 60- or 160-mm dishes coated with 10 mg/ml of poly-L-lysine. Cells were cultured for 8 days at 37°C in an atmosphere of 5% CO2/95% air in a Basal Eagle’s Medium supplemented with 10% heat-inactivated fetal calf serum, 25 mM KCl, 2 mM glutamine, and 100 mg/ml gentamicin. Cytosine-b-arabinofuranoside (10 mM) was added 24 h after plating to limit the number of nonneuronal cells. All experiments were approved by the local Ethical Committee for animal experiments.
2.2. Cell exposure to PDC Each culture of cerebellar granule cells was divided into control and PDC groups. PDC (from 10 mM stock solution in water) was added once, 12–48 h before RNA extraction and sample preparation of proteins or fixation of cultures for immunocytochemistry, to produce a final concentration of 100 mM. Medium samples for reverse-phase HPLC measurements of glutamate content were taken at various time points following PDC application.
2.3. Re6erse transcription-polymerase chain reaction Total RNA was extracted from one 160 mm culture dish per treatment group by using Ultraspec II RNA isolation system (Biotecx, TX) according to manufacturers instructions and as previously described (Cebere et al., 1999). Total RNA samples were transferred to clean tubes and stored at − 80°C until assayed. From each sample, a 5 ml aliquot was taken for spectrophotometric measurements of RNA concentration. Reverse transcription was combined with PCR to produce complementary DNA (cDNA) from the total RNA samples. Two microliter of total RNA from each cell extract were reverse transcribed using a random hexamer primer (pd (N)6; Pharmacia Biotech, Uppsala, Sweden) and 200 U M-MLV reverse transcriptase (Promega, WT) in a 25 ml reaction. Resulting cDNA samples were brought to 55 ml with DEPC-treated water. Intron-spanning gene-specific primer pairs for NMDA receptor subunits NR1C1, NR1N1, NR2A and NR2B were purchased from Stratagene (CA, USA). Primer-pair for rat b-actin, generating a 764-bp fragment, was purchased from Clontech (CA, USA). PCR was performed with 10 ml of diluted cDNA in a 25 ml reaction containing 2.5 ml 10 ×PCR buffer, 0.25 U Taq DNA polymerase, 1.5 mM MgCl2, 0.5 ml 10 mM dNTP mix and 1 mM each of sense and antisense primers for either the respective NMDA receptor subunit or bactin. Reaction mixtures were overlaid with mineral oil and 20 PCR cycles were performed (1 min at 94°C, 1 min at 62°C and 1 min at 72°C, with final extension of 10 min at 72°C). Aliquots (5 ml) of the reaction products were run on 2% agarose gels containing ethidium
G. Cebers et al. / Neurochemistry International 39 (2001) 151–160
bromide (0.5 mg/ml), to mark and visualise the PCR products. Gels were then photographed under UV light and negatives analysed densitometrically. NMDA subunit-specific primer-generated PCR product band intensities were normalised using values from PCR products generated by b-actin primers from the same cDNA batch.
2.4. Immunoblot analysis Immunoblot analysis of NMDA receptor subunit protein expression was carried out as previously described (Cebere et al., 1999; Cebers et al., 1999) with some modifications. Cells from one 160-mm culture dish (per group) were harvested into 1 ml of ice-cold phosphate-buffered saline (pH 7.4). Following centrifugation (15 min, 10000×g, 4°C) cells were homogenised in 10 volumes of Tris– HCl (5 mM), EDTA (5 mM), and phenylmethylsulphonyl fluoride (0.1 mM), and recentrifuged (10 min, 4000× g, 4°C). Supernatants were collected and centrifuged again (15 min, 15000× g, 4°C). Pellets, containing the membrane fraction of cellular proteins, were diluted in Tris– HCl (100 mM, pH 6.8), dithiothreitol (200 mM), sodium dodecyl sulfate (4%), bromophenol blue (0.2%), glycerol (20%), and b-mercaptoethanol (10%), boiled for 3 min, loaded (40 mg/lane) on 7% acrylamide/0.24% bisacrylamide resolving gel, and electrophoresed at 100 V for 2 h. Proteins were then transferred to PolyScreen polyvinylidene difluoride (PVDF) membranes (DuMedical Scandinavia, Stockholm, Sweden) and incubated for 2 h in Blotto containing 5% dry milk in Tris-buffered saline/Tween 20 (TBS-T; Tris, 10 mM; NaCl, 150 mM; Tween 20, 0.05%). Membranes were then incubated for 2 h in a Blotto solution of goat polyclonal antibody against NR1, NR2A, NR2B or NR2C subunit protein (1:1500; Santa Cruz Biotechnology, Santa Cruz, CA), washed (5 × 5 min) in TBS-T, and incubated for 1 h in Blotto solution of anti-goat horseradish peroxidase-conjugated secondary antibody (1:5000; Santa Cruz Biotechnology). Finally, membranes were washed (5× 5 min) in TBS-T, incubated in substrate solution (Renaissance Chemiluminescence Reagent Plus; DuMedical Scandinavia) for 1 min, and exposed to X-ray film. Horseradish peroxidase converts the substrate into its oxidised form, which is fluorescent. Quantity of the emitted fluorescence (registered by the X-ray film) from any given point on the PVDF membrane is proportional to the amount of the target protein (antigen) bound there. Levels of immunoreactivity were quantified densitometrically.
2.5. Immunocytochemistry For immunocytochemistry, cultures were grown on round glass coverslips (¥ 13 mm) in 24-well culture
153
plates. On DIV 8, at the end of PDC-treatment (100 mM for 48 h) cultures were washed three times with Dulbecco’s phosphate-buffered saline (DPBS; pH 7.5) supplemented with Mg2 + (0.5 mM) and Ca2 + (0.9 mM), then fixed in 4% paraformaldehyde for 20 min at room temperature (RT). After washing three times with DPBS, the coverslips carrying paraformaldehyde-fixed cells were incubated with 0.1% Triton X-100 (in DPBS) for 5 min at RT to permeabilise the cell membrane. Following three washes with DPBS, the unspecific binding sites were blocked with 2% bovine serum albumin (BSA; in DPBS) for 1 h at RT. Coverslips were then incubated with affinity purified rabbit anti-NR1 antibody (against all splice variants; Chemicon; 2 mg/ml in DPBS-2% BSA) for 1 h at 37°C. Then coverslips were washed 3 times with DPBS, 10 min each, and incubated with Alexa Fluor 488-labeled goat anti-rabbit IgG (Molecular Probes Europe, Leiden, The Netherlands; 10 mg/ml in DPBS-2% BSA) for 1 h at RT. After the final washing step (3 times with DPBS, 10 min each), coverslips were mounted in ProLong anti-fade mounting medium (Molecular Probes) and examined using a Leica DC 200 CCD camera fit on a Leica IRBE microscope equipped with 100× oil immersion objective, with a mercury lamp for wide-band excitation, and a set of emission band-pass filters. Fluorescence images were paired with differential interference contrast (DIC) images collected from the same fields of vision. Intensity of NR1-specific labelling in cell bodies and neurites of control or PDC-treated cells was quantified densitometrically by SigmaScanPro software (Jandel GmbH, Erkrath, Germany). Images were prepared for printing using CorelDraw software.
2.6. Materials Culture dishes were from Nunc (Roskilde, Denmark). Foetal calf serum, gentamicin and Eagle’s medium were from Life Technologies (Paisley, Scotland). PDC was from Tocris (Bristol, UK). All other chemicals were purchased from Sigma (St. Louis, MO).
3. Results
3.1. PDC-induced accumulation of glutamate PDC (100 mM) was added to the culture medium for 12, 24 or 48 h. In control cultures the mean glutamate concentration was 1.39 0.1 mM. PDC exposure increased the medium concentration of glutamate in a time-dependent manner: 37.49 2.9 mM at 12 h; 52.19 7.6 mM at 24 h; and 101.996.6 mM at 48 h (a more detailed time course of PDC-induced glutamate accumulation is shown in Cebers et al. (1999)).
154
G. Cebers et al. / Neurochemistry International 39 (2001) 151–160
3.2. Relati6e abundance of NR mRNAs in control and PDC-treated cells A representative RT-PCR analysis of NR1N1, NR1C1, NR2A, and NR2B mRNA expression in cerebellar granule cells grown in the absence or presence of 100 mM PDC is shown in Fig. 1. Amplification products with the gene-specific primers for the NMDA receptor subunits had expected sizes for all primer-pairs tested. For NR1, amplification with primers flanking the C1 or N1 splice-variant exon from rat resulted in two products (Fig. 1) corresponding to mRNAs either containing or lacking these exons. The respective exonpositive bands had larger molecular size (NR1C1 – 214 or 103 bp fragment in the presence or absence of the C1 exon; NR1N1 – 235 or 172 bp fragment in the presence or absence of the N1 exon). In both control and PDC-treated cells, the amount of N1-positive NR1 mRNA was too low for reliable densitometric analysis. Therefore, we were not able to analyse statistically the expression of N1-positive splice variants. However, based on visual assessment of the PCR product electrophoresis, PDC treatment did not seem to alter the abundance of N1- positive splice variants. In control cultures, the expression of C1-positive NR1 splice variants was about twice as abundant compared to C1-negative mRNAs (Fig. 1). However, 48 h following the PDC exposure the C1-positive splice variant expression was reduced by about 35% whereas, the expression of C1-negative NR1 mRNAs showed a tendency to increase (Fig. 2, panel A). Although this PDC effect on C1-negative splice variant expression was not statistically significant, the ratio of C1-positive/C1-negative, that in control cells was above 2, was reduced in PDC-exposed cells to 1.2 (Fig. 2, panel B). As demonstrated in Fig. 2, panel C, expression of N1-negative splice variants also seemed to decline following PDC treatment, but the effect was not significant (F = 1.3; P = 0.28; one-way ANOVA). These results indicate that although the PDC exposure changed the relative abundance of NR1 splice-variant expression, it did not cause an overall reduction of NR1 mRNA transcription. The sizes of NR2A- and NR2B-specific products were 207
and 183 bp, respectively. As can be seen from data presented in both Fig. 1 and Fig. 3, PDC treatment (100 mM; 12 –48 h) clearly reduced the expression of NR2 subunits. Compared to control cells, the amounts of both NR2A and NR2B (Fig. 3) were about 40–50% lower in PDC-treated cells. In both cases, the most prominent reduction of the respective mRNAs was achieved 48 h following the PDC addition to the incubation medium Fig. 3.
3.3. Immunoblot e6aluation of NR protein expression in membrane fractions of control and PDC-treated cells The relative amounts of mRNAs transcribed from a given gene are only suggestive of the proportions of their corresponding proteins. Therefore, the expression of NMDA receptor subunits was further examined by immunoblot analysis. The uniformity of protein transfer to the PVDF membranes was verified by Coomassie Brilliant Blue protein staining. As depicted in Fig. 5, there was no apparent difference in the overall pattern of protein electrophoresis between the control and PDC-exposed cells. In control cells, the immunoreactivities for all of the studied NMDA receptor subunit proteins, NR1, NR2A, NR2B and NR2C, were detected (Fig. 4). All subunit proteins (specific bands are indicated by arrows) had the expected size. The major immunoreactive band for NR2C was accompanied by lower molecular weight bands presumably consisting of NR2C breakdown products. The NR1 subunits were the most abundantly expressed, followed by NR2A and NR2B, whereas the expression of NR2C was considerably lower. It should be pointed out, however, that a direct comparison between immunoreactivity intensities of different antibody–antigen complexes is rather arbitrary, even though the same dilution was used for all antibodies, because of the differences in antibody reactivity and other intrinsic properties. The 48 h PDC exposure resulted in reduced intensity for the major immunoreactive band of anti-NR1 and all anti-NR2 staining. This PDC-induced reduction was clear and reproducible, and rather prominent already following 24 h PDC incubation (data not shown). The changes in
Fig. 1. A representative RT-PCR analysis of NR1N1, NR1C1, NR2A, and NR2B mRNA expression. Duplicate cDNA samples from different 8-day-old cerebellar granule cell culture preparations grown for the last 48 h in the presence or absence of 100 mM PDC were amplified using the corresponding primer pair. NR1C1 and NR1N1 primer-amplified PCR products separate as two bands representing C1 - or N1 - positive and negative splice variants of NR1 mRNAs. The lanes marked M contained 123-bp molecular weight standards. Typical results are shown for assays repeated 7 – 9 times.
G. Cebers et al. / Neurochemistry International 39 (2001) 151–160
155
treated samples were reduced by more than 60% relative to control cells. Similar to data from RT-PCR experiments, NR2A and NR2B proteins were reduced to a greater extent than NR1. However, the overall decrease in the expression of NMDA receptor subunit proteins appeared to be considerably stronger than the reduction of the corresponding mRNAs, suggesting an involvement of a post-transcriptional or post-translational regulation.
3.4. NR1 subunit protein expression in situ As shown in Fig. 6, NR1 subunit proteins were expressed on both cell bodies and neurites. Specific staining is seen as bright dots on the background of unspecific dark-green neuron outlines (panels A and B). In cell cultures pre-exposed for 48 h to PDC, the number as well as the intensity of these NR1 receptor clusters was lower than in control sister-cultures. Panels E and F show magnifications of single neurites from control (E) and PDC-exposed (F) cultures. These highmagnification images reveal a clear reduction of NR1
Fig. 2. Densitometric analysis of NR1C1 and NR1N1 splice variant mRNA expression in cerebellar granule cell cultures grown for the last 12 – 48 h in the absence or presence of 100 mM PDC. (A) PDC exposure produced a significant time-dependent decrease (**PB0.01, as compared to control) of NR1C1 expression. (B) The ratio between C1 -positive and C1 -negative NR1 subunits was reduced depending on the period of PDC exposure and was significantly lower that in control cells following a 48 h incubation (*PB 0.05, compared to control cells, Bonferroni’s test of multiple comparisons). (C) Expression of NR1N1 was not significantly changed by PDC exposure. Data are means 9SEM, derived from 5 –7 different PDC exposure experiments and are normalised to the amount of b-actin PCR product in each cDNA batch and the amount of respective mRNA in control cells ( =100%).
immunostaining induced by PDC exposure were quantified as described under Section 2. These results are presented in the bottom panel of Fig. 4. PDC treatment (48 h, 100 mM) diminished the anti-NR1 immunoreactivity by 55%. In comparison, both antiNR2A and anti-NR2B immunoreactivities were reduced by approximately 80%. For anti-NR2C immunoreactivity, the specific 190-kDa bands in PDC-
Fig. 3. Densitometric analysis of RT-PCR measurement of NR2A and NR2B subunit mRNA expression in cerebellar granule cell cultures grown for the last 12 – 48 h in the absence or presence of 100 mM PDC. Data are means 9 SEM, derived from 5 – 7 different PDC exposure experiments and are normalised to the amount of b-actin PCR product in each cDNA batch and the amount of respective mRNA in control cells ( =100%). *PB 0.05; **PB0.01, different from control, as determined by Bonferroni multiple comparison test.
156
G. Cebers et al. / Neurochemistry International 39 (2001) 151–160
and 45% as measured in cell bodies and dendrites, respectively. Furthermore, as can be seen from panels C and D of Fig. 6, there was no obvious difference in the morphology of PDC-exposed neurons as compared to the intact culture, thereby confirming our previous observation that the majority of cerebellar granule cells were able to survive and adjust to the increasing medium glutamate concentrations (Cebers et al., 1999).
4. Discussion The main finding of this study is that prolonged blockade of glutamate reuptake, which produced a linear increase in the medium concentration of glutamate (from 1 mM to about 100 mM during the 48 h PDC exposure), substantially reduced the expression of NMDA receptor subunits at both mRNA and protein levels. These results provide an action mechanism for the down-regulation of NMDA receptor-mediated functional responses observed following the prolonged glutamate reuptake blockade in cerebellar granule cell culture (Cebers et al., 1999). Both the functional activity and the number of NMDA receptors appears to be regulated by an activity-dependent mechanism, at least in primary neuronal cultures (Fields and Nelson, 1992; Didier et al., 1994; Resink et al., 1995). Convincing experimental evidence indicates that the responsiveness and/or the number of NMDA receptors increase following chronic treatment with NMDA receptor antagonists (O’Brien and Fishbach, 1986; McDonald et al., 1990; Williams et al., 1992; Didier et al., 1994; van den Pol et al., 1996; Rao and Craig, 1997). Studies addressing effects of chronic NMDA receptor stimulation are, however, rather few, Fig. 4. Immunoblot analysis of the NR1, NR2A, NR2B and NR2C receptor subunit protein expression in cerebellar granule cells grown for the last 48 h in the absence or presence of 100 mM PDC. Antibody binding was visualised by chemiluminescence. All subunit proteins (the specific bands are indicated by arrows) had the expected size. The positions of molecular mass standards (kDa) are shown on the left. The bottom panel shows results of the densitometric analysis of replicated immunoblots similar to these presented in the upper panels. Data are means 9 SEM, derived from 3 –7 different PDC exposure experiments and are normalised to the amount of respective receptor protein expressed in control cells ( = 100%) from the sister cultures. **PB 0.01, as compared to control cultures, Bonferroni test of multiple comparisons.
protein expression on the neurites of PDC-exposed granule cells. Densitometric measurements (bar-figure below the images) from a large number of cells (260– 340 cells per condition) confirmed a highly significant reduction of NR1 immunostaining in both cell bodies and dendritic network of PDC-exposed cells. Thus, the expression of NR1 protein was reduced by about 30%
Fig. 5. The uniformity of protein transfer following electrophoretic separation to the PVDF membranes was confirmed by total protein staining. Aliquots containing 40 mg protein from the membrane fraction were subjected to 7% SDS-PAGE and transferred to the PVDF membrane. After the immunochemical procedures were completed membranes were always stained with Coomassie Brilliant Blue.
G. Cebers et al. / Neurochemistry International 39 (2001) 151–160
157
Fig. 6. Localisation of NR1 receptor subunit immunoreactivity in control (A, C, E) and PDC- exposed (B, D, F) cerebellar granule cells. Panels A and B show the NR1-specific immunofluorescent staining in representative fields of vision using a 100 × objective. For better assessment of cellular morphology the same fields of vision were photographed in transparent mode using a DIC filter (panels C and D). Captures of neurite sections (white rectangles in panels A and B) are shown at 4 × zoom in panels E and F. Figure below images shows the results of densitometric analysis of the intensity of NR1-specif fluorescence measured from cell bodies or dendritic network. 260 – 340 cells per condition were analysed. ***PB0.001, as compared to control cells; Bonferroni test for multiple comparisons.
158
G. Cebers et al. / Neurochemistry International 39 (2001) 151–160
probably because of the obvious difficulty to balance a sufficient stimulatory effect at the NMDA receptor with an intact neuronal viability. Nevertheless, existing studies show a clear tendency towards a reduction of NMDA receptor-mediated responses following prolonged stimulation. Thus, Debski et al. (1991) reported a decrease in the sensitivity to applied NMDA following chronic treatment with NMDA in frog tecta. Likewise, the ability of a prolonged NMDA receptor activation to cause NMDA receptor down-regulation has been confirmed using other experimental models (Chuang et al., 1992; Oster and Schramm, 1993; Didier et al., 1994; Resink et al., 1995; Dickie et al., 1996). Those studies are in full agreement with our present and past (Cebers et al., 1999) results, which show that cerebellar granule neurons can effectively counteract the excitotoxic effects of glutamate by altering the expression and/or sensitivity of the NMDA receptors. As already mentioned, studies on activity-dependent regulation of NMDA receptor expression and functions have been hampered by the problem of delivering sustained NMDA receptor stimulation without causing neuronal death. It seems that a necessary condition here is that the level of NMDA receptor stimulation is not increased abruptly as, for instance, by applying buffers containing high concentrations of glutamate. In this respect, our model of glutamate reuptake inhibition offers a considerable advantage, in that it allows a linear and continuous accumulation of glutamate causing an increasing activation of NMDA receptors without producing concomitant excitotoxicity, at least not in cerebellar granule cells. Another advantage of our method is that in variance with the traditional application of exogenous NMDA receptor agonists, glutamate is released endogenously at the established glutamatergic synapses. This condition may be especially important in the beginning of adaptative processes, when the overall medium levels of glutamate are still low, but, at the same time, glutamate concentration in the vicinity of the synapse, and therefore, the level of glutamate receptor activation (Diamond and Jahr, 1997; Mennerick et al., 1999), is increased from the very beginning of the transporter blockade. Indeed, our previous studies showed that already one hour of PDC incubation was sufficient for NMDA receptor-mediated enhancement of the activator protein-1 (AP-1) DNA-binding activity (Kova´ cs et al., 1999), an event that may directly affect the transcriptional regulation of NMDA receptor expression (Bai and Kusiak, 1993). Our present data (especially from Section 3.3) suggest that changes in the relative abundance of various NR1 splice variants and NR2 subunits following PDC treatment may be at least partially responsible for the observed reduction of NMDA receptor-mediated functional effects (Cebers et al., 1999), thus supporting the view that the subunit composition of the NMDA recep-
tor complex is of crucial importance for defining the functional properties of this receptor (Kutsuwada et al., 1992; Watanabe et al., 1992; Durand et al., 1993; Hollmann et al., 1993; Buller et al., 1994; Feldmeyer and Cull-Candy, 1996). The NMDA receptor is a heteromeric complex comprised of a mandatory NR1 subunit and one or several NR2A–D subunits (Nakanishi et al., 1990; Moriyoshi et al., 1991; Sugihara et al., 1992; Durand et al., 1993; Forrest et al., 1994; Laurie and Seeburg, 1994). NR1 protein, the main NMDA receptor subunit, is encoded by a gene with 22 exons, and at least 8 splice variants have been identified derived from one alternatively spliced cassette at the N-terminal region (N1 exon) and two at the C-terminal region (C1 and C2 exon) (Hollmann et al., 1993). In our experiments, the expression of the C1-positive NR1 splice-variant was reduced by about 40% in PDC-exposed cultures, whereas the expression of C1-negative mRNA was not significantly changed. Therefore, the ratio of C1-positive/C1-negative NR1 was reduced nearly by half in PDC-exposed cells, indicating a major re-arrangement in relative abundance of NR1 splice variants. It is notable that C1 exon contains target sites for protein kinase C, as well as a binding site for calmodulin (Tingley et al., 1993; Hisatune et al., 1997), which inactivates NMDA channels in a Ca2 + - and phosphorylation-dependent manner (Ehlers et al., 1996; Hisatune et al., 1997). Moreover, C1 exon is also involved in the NR1 interaction with the cytoskeleton (Wyszynski et al., 1997) and anchoring proteins like PSD-95 (Kornau et al., 1995), which may be important for the subcellular localisation of the NR1 subunit and its ability to assemble into receptor clusters at the post-synaptic membrane (Tingley et al., 1993; Ehlers et al., 1996; Okabe et al., 1999). Indeed, it has been shown that the cell membrane expression of especially NR1 but also NR2 subunits may involve such regulatory steps as surface trafficking and phosphorylation of the intracellular domains on the NMDA receptors (Hall and Soderling, 1997). As a result of such regulation, NMDA receptors may be trafficked with differential efficiency and exhibit different levels of phosphorylation which can affect the distribution of NR1 subunits within the plasma membrane (Ehlers et al., 1995). The expression of N1-negative NR1 splice-variants, however, did not change following the PDC treatment, indicating that PDC exposure did not cause an overall reduction of NR1 mRNA transcription. This observation is surprising because the reduction of NR1 subunit proteins following the PDC treatment was clearly detected both in the immunoblot and the immunocytochemistry assays. Notably, Didier et al. (1994), who prepared their cerebellar granule cells in low K+ conditions in the presence of NMDA, also failed to observe a good correlation between the considerable decrease of NMDA receptor sensitivity (accompanied by reduced
G. Cebers et al. / Neurochemistry International 39 (2001) 151–160
binding of [3H]MK-801) and the expression of mRNA for the NR1 subunit. Therefore, it seems reasonable to suggest that NR1 mRNA could be modulated posttranscriptionally in a manner that changes its translational efficiency. In fact, modulation of NMDA receptor activity through the control of subunit translation has already been proposed for the NR2A subunit (Wood et al., 1996). PDC exposure reduced the expression of NR1 subunit protein only by half although our previous functional experiments displayed an almost complete lack of response to glutamate in equally treated cultures (Cebers et al., 1999). This discrepancy could be explained by the fact that the presence of a NR2 subunit is required for insertion of NR1 into the cell membrane (McIlhinney et al., 1996). Thus, it is conceivable that the dramatic reduction of NR2 subunits observed following the PDC exposure may have precluded the membrane trafficking of the remaining NR1 subunit proteins. Taken together our current data show that a prolonged blockade of glutamate reuptake produced a linear increase in medium glutamate concentration, which suppressed the expression of NMDA receptor subunits at both mRNA and protein levels. However, the decrease of the membrane-bound receptor proteins was considerably greater than the reduction of the corresponding mRNAs, thus suggesting that post-transcriptional or post-translational regulation may be involved. The fact that PDC exposure also changed the relative abundances of mRNAs for several NR1 splicevariants suggests not only that the total numbers of NMDA receptor subunits were reduced, but also that the remaining NMDA receptors may have changed their subunit composition followed by altered functional properties. These results support our earlier findings indicating that both the functional activity and the number of NMDA receptors, at least in culture conditions, are effectively regulated in an activity-dependent manner. Although, the exact mechanism of this phenomenon remains to be elucidated, the PDC-induced down-regulation of NMDA receptors in primary neuronal cultures provides a powerful experimental tool to study the regulation of NMDA receptor expression.
Acknowledgements G.C. was a recipient of a post-doc fellowship from the Swedish Brain Foundation. A.D.K. was a recipient of a Visiting Scientist Fellowship from the Wenner– Gren Foundations. This study was supported by the Swedish Medical Research Council (project no. 7688), the Swedish Society for Medical Research, A, ke Wiberg’s Foundation, Lars Hierta’s Foundation, Loo and Hans Osterman’s Foundation, Go¨ sta Fraenckel’s
159
Foundation, Sigurd and Elsa Golje’s Foundation, Kapten Artur Eriksson’s Foundation, and funds from the Karolinska Institutet.
References Bai, G., Kusiak, J.W., 1993. Cloning and analysis of the 5% flanking sequence of the rat N-methyl-D-aspartate receptor 1 (NMDAR1) gene. Biochemica Biophysica Acta 1152, 197 – 200. Bliss, T.V.P., Collingridge, G.L., 1993. A synaptic model of memory: long-term potentiation in the hippocampus. Nature 361, 31 –39. Boulter, J., Hollmann, M., O’Shea-Greenfield, A., Hartley, M., Deneris, E., Maron, C., Heinemann, S., 1990. Molecular cloning and functional expression of glutamate receptor subunit genes. Science 249, 1033 – 1037. Buller, A.L., Larson, H.C., Schneider, B.E., Beaton, J.A., Morrisett, R.A., Monaghan, D.T., 1994. The molecular basis of NMDA receptor subtypes: native receptor diversity is predicted by subunit composition. J. Neurosci. 14, 5471 – 5484. Burgoyne, R.D., Cambray-Deakin, M.A., 1988. The cellular neurobiology of neuronal development: the cerebellar granule cell. Brain Res. Rev. 13, 77 – 101. Burgoyne, R.D., Pearce, I.A., Cambray-Deakin, M.A., 1988. NMethyl-D-aspartate raises cytosolic calcium concentration in rat cerebellar granule cells. Neurosci. Lett. 91, 47 – 52. Cebere, A., Cebers, G., Liljequist, S., 1999. Enhancement of NMDAinduced functional responses without concomitant NMDA receptor changes following chronic ethanol exposure in cerebellar granule cells. Naunyn-Schmiedeberg’s Arch. Pharmacol. 360, 623 – 632. Cebers, G., Liljequist, S., 1995. Modulation of AMPA/kainate receptors by cyclothiazide increases cytoplasmic free Ca2 + uptake and 45 Ca2 + in brain neurons. Eur. J. Pharmacol. Mol. Pharmacol. Sec. 290, 105 – 115. Cebers, G., Cebere, A., Wa¨ gner, A., Liljequist, S., 1999. Prolonged inhibition of glutamate reuptake down-regulates NMDA receptor functions in cultured cerebellar granule cells. J. Neurochem. 72, 2181 – 2190. Choi, D.W., 1992. Excitotoxic cell death. J. Neurobiol. 23, 1261 – 1276. Chuang, D.M., Gao, X.M., Paul, S.M., 1992. N-methyl-D-aspartate exposure blocks glutamate toxicity in cultured cerebellar granule cells. Mol. Pharmacol. 42, 210 – 216. Collingridge, G.L., Lester, R.A., 1989. Excitatory amino acid receptors in the vertebrate central nervous system. Pharmacol. Rev. 40, 144 – 209. Collingridge, G.L., Singer, W., 1990. Excitatory amino acid receptors and synaptic plasticity. Trends in Pharmacological Sciences 11, 290 – 296. Debski, E.A., Cline, H.T., McDonald, J.W., Constantine-Paton, M., 1991. Chronic application of NMDA decreases the NMDA sensitivity of the evoked tectal potential in the frog. J. Neurosci. 11, 2947 – 2957. Diamond, J.S., Jahr, C.E., 1997. Transporters buffer synaptically released glutamate on a submillisecond time scale. J. Neurosci. 17, 4672 – 4687. Dickie, B.G.M., Holmes, C., Greenfield, S.A., 1996. Neurotoxic and neurotrophic effects of chronic N-methy-D-aspartate exposure upon mesencephalic dopaminergic neurons in organotypic culture. Neuroscience 72, 731 – 741. Didier, M., Mienville, J.-M., Soubrie`, P., Bockaert, J., Berman, S., Bursztajn, S., Pin, J.-P., 1994. Plasticity of NMDA receptor expression during mouse cerebellar granule cell development. Eur. J. Neurosci. 6, 1536 – 1543.
160
G. Cebers et al. / Neurochemistry International 39 (2001) 151–160
Durand, G.M., Bennett, M.V.L., Zukin, S., 1993. Splice variants of the N-methyl-D-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C. Proc. Natl. Acad. Sci. USA 90, 6731 –6735. Ehlers, M.D., Tingley, W.G., Huganir, R.L., 1995. Regulated subcellular distribution of the NR1 subunit of the NMDA receptor. Science 269, 1734 – 1737. Ehlers, M.D., Zhang, S., Bernhardt, J.P., Huganir, R.L., 1996. Inactivation of NMDA receptors by direct interaction of calmodulin with the NR1 subunit. Cell 84, 745 –755. Feldmeyer, D., Cull-Candy, S., 1996. Functional consequences of changes in NMDA receptor subunit expression during development. J. Neurocytol. 25, 857 –867. Fields, R.D., Nelson, P.G., 1992. Activity-dependent development of the vertebrate central nervous system. Inter. Rev. Neurobiol. 34, 133– 214. Forrest, D., Yuzaki, M., Soares, H.D., Ng, L., Luk, D.C., Sheng, M., Stewart, C.L., Morgan, G.I., Connor, G.A., Curran, T., 1994. Targeted disruption of NMDA receptor 1 gene abolishes NMDA responses and results in neonatal death. Neuron 13, 325– 328. Gjertsen, B.T., Doskeland, S.O., 1995. Protein phosphorylation in apoptosis. Biochemica Biophysica Acta 1269, 187 –199. Hall, R.A., Soderling, T.R., 1997. Differential surface expression and phosphorylation of the N-methyl-D-aspartate receptor subunits NR1 and NR2 in cultured hippocampal neurons. J. Biol. Chem. 272, 4135 – 4140. Hisatune, C., Umemori, H., Inoue, T., Michikawa, T., Kohda, K., Mikoshiba, K., Yamamoto, T., 1997. Phosphorylation-dependent regulation of N-methyl-D-aspartate receptors by calmodulin. J. Biol. Chem. 272, 20805 –20810. Hollmann, M., Boulter, J., Maron, C., Beasley, L., Sullivan, J., Pecht, G., Heinemann, S., 1993. Zink potentiates agonist-induced currents at certain splice variants of the NMDA receptor. Neuron 10, 943 – 954. Hollmann, M., Heinemann, S., 1994. Cloned glutamate receptors. Ann. Rev. Neurosci. 17, 31 –108. Kornau, H.-C., Schenker, L.T., Kennedy, M.B., Seeburg, P.H., 1995. Domain interaction between NMDA receptor subunits and the postsynaptic density protein PSD-95. Science 269, 1737 –1740. Kova´ cs, A.D., Cebers, G., Liljequist, S., 1999. Prolonged enhancement of AP-1 DNA-binding by blockade of glutamate uptake in cultured neurons. Neuroreport 10, 1805 –1809. Kutsuwada, T., Kashiwabuchi, N., Mori, H., Sakimura, K., Kushiya, E., Araki, K., Meguro, H., Masaki, H., Kumanishi, T., Arakawa, M., Mishina, M., 1992. Molecular diversity of the NMDA receptor channel. Nature 358, 36 –41. Laurie, D.J., Seeburg, P.H., 1994. Regional and developmental heterogenity in splicing of the rat brain NMDAR1 mRNA. J. Neurosci. 14, 3180 – 3194. Mayer, J.I., Westbrook, G.L., 1987. The physiology of excitatory amino acids in the vertebrate central nervous system. Prog. Neurobiol. 28, 197 – 276. McBean, G.J., Roberts, P.J., 1985. Neurotoxicity of L-glutamate and DL-threo-3-hydroxyaspartate in the rat striatum. J. Neurochem. 44, 247 – 254. McDonald, J.W., Silverstein, F.S., Johnson, M.V., 1990. MK-801 pretreatment enhances N-methyl-D-aspartate-mediated brain injury and increases brain N-methyl-D-aspartate recognition site binding in rats. Neuroscience 38, 103 –113. McIlhinney, R.A.J., Molnar, E., Atack, J.R., Whiting, P.J., 1996. Cell surface expression of the human N-methyl-D-aspartate receptor subunit 1a requires the co-expression of the NR2a subunit in transfected cells. Neuroscience 70, 989 –997. Meldrum, B., Garthwaite, J., 1990. Excitatory amino acid neurotoxicity and neurodegenerative diseases. Trends in Pharmacological Sciences 11, 379 – 387. .
Mennerick, S., Shen, W., Xu, W., Benz, A., Tanaka, K., Shimamoto, K., Isenberg, K.E., Krause, J.E., Zorumski, C.F., 1999. Substrate turnover by transporters curtails synaptic glutamate transients. J. Neurosci. 19, 9242 – 9251. Monaghan, D.T., Yao, D., Cotman, C.W., 1985. L-[3H]Glutamate binds to kainate-, NMDA-, and AMPA-sensitive binding sites: an autoradiographic analysis. Brain Res. 340, 378 – 383. Moriyoshi, K., Masu, M., Ishii, T., Shigemoto, R., Mizuno, N., Nakanishi, S., 1991. Molecular cloning and characterization of the rat NMDA receptor. Nature 354, 31 – 35. Nakanishi, N., Shneider, N.A., Axel, R., 1990. A family of glutamate receptor genes: evidence for the formation of heteromultimeric receptors with distinct channel properties. Neuron 5, 569 – 581. Nicoll, R.A., Kauer, J.A., Malenka, R.C., 1988. The current excitement in long-term potentiation. Neuron 1, 97 – 103. O’Brien, R.J., Fishbach, G.D., 1986. Modulation of embryonic chick motorneuron glutamate sensitivity by interneurons and agonists. J. Neurosci. 6, 3290 – 3296. Okabe, S., Miwa, A., Okado, H., 1999. Alternative splicing of the C-terminal domain regulates cell surface expression of the NMDA receptor NR1 subunit. J. Neurosci. 19, 7781 – 7792. Orrenius, S., Nicotera, P., 1993. The calcium ion and cell death. J. Neural Transm. 43, 1 – 11. Oster, Y., Schramm, M., 1993. Down-regulation of NMDA receptor activity by NMDA. Neurosci. Lett. 163, 85 – 88. Rao, A., Craig, A.M., 1997. Activity regulates the synaptic localisation of the NMDA receptor in hippocampal neurons. Neuron 19, 801 – 812. Resink, A., Villa, M., Benke, D., Mohler, H., Balazs, R., 1995. Regulation of the expression of NMDA receptor subunits in rat cerebellar granule cells: effect of chronic K +-induced depolarization and NMDA exposure. J. Neurochem. 64, 558 – 565. Schoepp, D.D., Conn, P.J., 1993. Metabotropic glutamate receptors in brain function and pathology. Trends in Pharmacological Sciences 141, 13 – 20. Sugihara, H., Moriyoshi, K., Ishii, T., Masu, M., Nakanishi, S., 1992. Stuctures and properties of seven isoforms of the NMDA receptor generated by alternative splicing. Biochem. Biophys. Res. Commun. 185, 826 – 832. Tanabe, Y., Masu, M., Ishii, T., Sigemoto, R., Nakanishi, S., 1992. A family of metabotropic glutamate receptors. Neuron 8, 169 – 179. Tingley, W.G., Roche, K.W., Thompson, A.K., Huganir, R.L., 1993. Regulation of NMDA receptor phosphorylation by alternative splicing of the C-terminal domain. Nature 364, 70 – 73. Turski, L., Turski, W.A., 1993. Towards an understanding of the role of glutamate in neurodegenerative disorders: energy metabolism and neuropathology. Experientia 49, 1064 – 1072. van den Pol, A.N., Obrietan, K., Belousov, A., 1996. Glutamate hyperexcitability and seizure-like activity throughout the brain and spinal cord upon relief from chronic glutamate receptor blockade in culture. Neuroscience 74, 653 – 674. Watanabe, M., Inoue, Y., Sakimura, K., Mishina, M., 1992. Developmental changes in distribution of NMDA-receptor channel subunit mRNAs. Neuroreport 3, 1138 – 1140. Williams, K., Dichter, M.A., Molinoff, P.B., 1992. Up-regulation of N-methyl-D-aspartate receptors on cultured cortical neurons after exposure to antagonists. Mol. Pharmacol. 42, 147 – 151. Wood, M.W., VanDongen, H.M., VanDongen, A.M., 1996. The 5%-untranslated region of the N-methyl-D-aspartate receptor NR2A subunit controls efficiency of translation. J. Biol. Chem. 271, 8115 – 8120. Wyszynski, M., Lin, J., Rao, A., Nigh, E., Beggs, A.H., Craig, A.M., 1997. Competitive binding of a-actinin and calmodulin to the NMDA receptor. Nature 385, 439 – 442.
